Combining anti-HLA-DR or anti-Trop-2 antibodies with microtubule inhibitors, PARP inhibitors, Bruton kinase inhibitors or phosphoinositide 3-kinase inhibitors significantly improves therapeutic outcome in cancer

ABSTRACT

The present invention relates to combination therapy with drugs, such as microtubule inhibitors, PARP inhibitors, Bruton kinase inhibitors or PI3K inhibitors, with antibodies or immunoconjugates against HLA-DR or Trop-2. Where immunoconjugates are used, they preferably incorporate SN-38 or pro-2PDOX. The immunoconjugate may be administered at a dosage of between 1 mg/kg and 18 mg/kg, preferably 4, 6, 8, 9, 10, 12, 16 or 18 mg/kg, more preferably 8 or 10 mg/kg. The combination therapy can reduce solid tumors in size, reduce or eliminate metastases and is effective to treat cancers resistant to standard therapies, such as radiation therapy, chemotherapy or immunotherapy. Preferably, the combination therapy has an additive effect on inhibiting tumor growth. Most preferably, the combination therapy has a synergistic effect on inhibiting tumor growth.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/618,317, filed Jun. 9, 2017, which was a divisional of U.S. patentapplication Ser. No. 15/190,805 (now issued U.S. Pat. No. 9,707,302),filed Jun. 23, 2016, which was a continuation-in-part of U.S. patentapplication Ser. No. 15/069,208 (now issued U.S. Pat. No. 10,137,196),filed Mar. 14, 2016, which was a continuation-in-part of U.S. patentapplication Ser. No. 14/667,982 (now issued U.S. Pat. No. 9,493,573),filed Mar. 25, 2015, which was a divisional of U.S. patent applicationSer. No. 13/948,732 (now issued U.S. Pat. No. 9,028,833), filed Jul. 23,2013. This application claims the benefit under 35 U.S.C. 119(e) of U.S.Provisional Patent Application 62/184,331, filed Jun. 25, 2015,62/201,361, filed Aug. 5, 2015, 62/250,715, filed Nov. 4, 2015, and62/263,134, filed Dec. 4, 2015. The entire text of each priorityapplication is incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 23, 2016, isnamed IMM365US1_SL.txt and is 61,107 bytes in size.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to therapeutic use of anti-TAA antibodiesor immunoconjugates, such as antibodies or immunoconjugates againstHLA-DR or Trop-2, in combination with one or more drugs, wherein thecombination therapy is more effective than the antibody alone, drugalone, or the combined effects of drug and antibody alone. In preferredembodiments, the combination exhibits synergistic effects. In otherpreferred embodiments, the drugs of use may be ones that induce DNAstrand breaks, such as auristatins, colicheamicins, camptothecins (e.g.,SN-38) or a prodrug form of 2-pyrrolinodoxorubicine (P2PDox). Exemplarydrugs inducing DNA strand breaks include, but are not limited to, SN-38,P2PDox, topotecan, doxorubicin, etoposide, cisplatinum, oxaliplatin, orcarboplatin. In alternative embodiments, the drugs of use forcombination therapy belong to the categories of microtubule inhibitors,PARP inhibitors, Bruton kinase inhibitors or phosphoinositide 3-kinase(PI3K) inhibitors. The drugs may be administered separately or togetherwith the antibodies, or may be conjugated to the antibody prior toadministration. In the latter case, the antibodies and drugs may belinked via an intracellularly-cleavable linkage that increasestherapeutic efficacy. In other alternative embodiments, the antibody maybe conjugated to a different drug (such as SN-38) to form animmunoconjugate, and the immunoconjugate may be administered incombination with with a microtubule inhibitor, PARP inhibitor, Brutonkinase inhibitor or PI3K inhibitor. Preferably, immunoconjugates areadministered at specific dosages and/or specific schedules ofadministration that optimize their therapeutic effect. The optimizeddosages and schedules of administration of antibody-drug conjugates(ADCs) for human therapeutic use disclosed herein show unexpectedsuperior efficacy that could not have been predicted from animal modelstudies, allowing effective treatment of cancers that are resistant tostandard anti-cancer therapies, including irinotecan (CPT-11),paclitaxel or other compounds that induce DNA strand breaks.Surprisingly, combination therapy with antibody-SN38 immunoconjugatesand microtubule inhibitors or PARP inhibitors shows unexpectedsynergistic effects. In a particularly preferred embodiment, an SN-38conjugated antibody of use is an anti-Trop-2 antibody, such as IMMU-132(sacitizumab govitecan, also known as hRS7-CL2A-SN-38 or IMMU-132). Morepreferably, the methods are of use in treating triple-negative breastcancer (TNBC), metastatic colorectal cancer, SCLC or NSCLC, as well asother Trop-2-expressing cancers. Other SN-38 conjugates, such as withantibodies targeting other cancer-associated antigens, such as CD19,CD20, CD22, CD66e (CEACAM5), CD74, HLA-DR, IGF-1R, folate receptor, andothers listed below, also can be synergistically effective, or at leastadditive without increasing dose-limiting toxicities, when combined withsimilar classes of drugs. In another preferred embodiment, ananti-HLA-DR antibody of use is a humanized L243 antibody (hL243).

Background of the Invention

For many years it has been an aim of scientists in the field ofspecifically targeted drug therapy to use monoclonal antibodies (MAbs)for the specific delivery of toxic agents to human cancers. Conjugatesof MAbs that target tumor-associated antigens (TAA) and suitable toxicagents have been developed, but have had mixed success in the therapy ofcancer in humans, and virtually no application in other diseases, suchas infectious and autoimmune diseases. The toxic agent is most commonlya chemotherapeutic drug, although particle-emitting radionuclides, orbacterial or plant toxins, have also been conjugated to MAbs, especiallyfor the therapy of cancer (Sharkey and Goldenberg, CA Cancer J Clin.2006 July-August; 56(4):226-243) and, more recently, withradioimmunoconjugates for the preclinical therapy of certain infectiousdiseases (Dadachova and Casadevall, Q J Nucl Med Mol Imaging 2006;50(3):193-204).

The advantages of using MAb-chemotherapeutic drug conjugates are that(a) the chemotherapeutic drug itself is structurally well defined; (b)the chemotherapeutic drug is linked to the MAb protein using verywell-defined conjugation chemistries, often at specific sites remotefrom the MAbs' antigen-binding regions; (c) MAb-chemotherapeutic drugconjugates can be made more reproducibly and usually with lessimmunogenicity than chemical conjugates involving MAbs and bacterial orplant toxins, and as such are more amenable to commercial developmentand regulatory approval; and (d) the MAb-chemotherapeutic drugconjugates are orders of magnitude less toxic systemically thanradionuclide MAb conjugates, particularly to the radiation-sensitivebone marrow.

Camptothecin (CPT) and its derivatives are a class of potent antitumoragents. Irinotecan (also referred to as CPT-11) and topotecan are CPTanalogs that are approved cancer therapeutics (Iyer and Ratain, CancerChemother. Phamacol. 42: S31-S43 (1998)). CPTs act by inhibitingtopoisomerase I enzyme by stabilizing topoisomerase I-DNA complex (Liu,et al. in The Camptothecins: Unfolding Their Anticancer Potential, LiehrJ. G., Giovanella, B. C. and Verschraegen (eds), NY Acad Sci., NY922:1-10 (2000)). CPTs present specific issues in the preparation ofconjugates. One issue is the insolubility of most CPT derivatives inaqueous buffers. Second, CPTs provide specific challenges for structuralmodification for conjugating to macromolecules. For instance, CPT itselfcontains only a tertiary hydroxyl group in ring-E. The hydroxylfunctional group in the case of CPT must be coupled to a linker suitablefor subsequent protein conjugation; and in potent CPT derivatives, suchas SN-38, the active metabolite of the chemotherapeutic CPT-11, andother C-10-hydroxyl-containing derivatives such as topotecan and10-hydroxy-CPT, the presence of a phenolic hydroxyl at the C-10 positioncomplicates the necessary C-20-hydroxyl derivatization. Third, thelability under physiological conditions of the δ-lactone moiety of theE-ring of camptothecins results in greatly reduced antitumor potency.Therefore, the conjugation protocol is performed such that it is carriedout at a pH of 7 or lower to avoid the lactone ring opening. However,conjugation of a bifunctional CPT possessing an amine-reactive groupsuch as an active ester would typically require a pH of 8 or greater.Fourth, an intracellularly-cleavable moiety preferably is incorporatedin the linker/spacer connecting the CPTs and the antibodies or otherbinding moieties.

A need exists for more effective methods of preparing and administeringantibody-drug conjugates, such as antibody-SN-38 conjugates, and moreeffective combination therapy with antibodies or immunoconjugates anddrugs, such as microtubule inhibitors, PARP inhibitors, Bruton kinaseinhibitors or PI3Ks inhibitors.

SUMMARY OF THE INVENTION

In preferred embodiments, the invention involves combination therapyusing an anti-HLA-DR or anti-Trop-2 antibody, or immunoconjugatesthereof, in combination with a drug selected from the group consistingof microtubule inhibitors, PARP inhibitors, Bruton kinase inhibitors orphosphoinositide 3-kinase (PI3K) inhibitors. More preferably, thecombination therapy is more effective than antibody or immunoconjugatealone, drug alone, or the sum of the effects of antibody orimmunoconjugate and drug. Most preferably, the combination exhibitssynergistic effects for treatment of diseases, such as cancer, in humansubjects. In embodiments involving use of immunoconjugates, the antibodyis preferably conjugated to a CPT moiety, such as SN-38, or to ananthracycline, such as pro-2PDOX.

As used herein, the abbreviation “CPT” may refer to camptothecin or anyof its derivatives, such as SN-38, unless expressly stated otherwise.The present invention provides improved methods and compositions forpreparing and administering CPT-antibody immunoconjugates. Preferably,the camptothecin is SN-38. The disclosed methods and compositions are ofuse for the treatment of a variety of diseases and conditions which arerefractory or less responsive to other forms of therapy, and can includediseases against which suitable antibodies or antigen-binding antibodyfragments for selective targeting can be developed, or are available orknown. Preferred diseases or conditions that may be treated with thesubject antibodies or immunoconjugates include, for example, cancer orautoimmune disease. Most preferably, the immunoconjugates are of use totreat cancer, particularly cancers that have proven refractory to priortreatment with standard cancer therapies.

Preferably, the targeting moiety is an antibody, antibody fragment,bispecific or other multivalent antibody, or other antibody-basedmolecule or compound. The antibody can be of various isotypes,preferably human IgG1, IgG2, IgG3 or IgG4, more preferably comprisinghuman IgG1 hinge and constant region sequences. The antibody or fragmentthereof can be a chimeric human-mouse, a chimeric human-primate, ahumanized (human framework and murine hypervariable (CDR) regions), orfully human antibody, as well as variations thereof, such as half-IgG4antibodies (referred to as “unibodies”), as described by van der NeutKolfschoten et al. (Science 2007; 317:1554-1557). More preferably, theantibody or fragment thereof may be designed or selected to comprisehuman constant region sequences that belong to specific allotypes, whichmay result in reduced immunogenicity when the antibody orimmunoconjugate is administered to a human subject. Preferred allotypesfor administration include a non-G1m1allotype (nG1m1), such as G1m3,G1m3,1, G1m3,2 or G1m3,1,2. More preferably, the allotype is selectedfrom the group consisting of the nG1m1, G1m3, nG1m1, 2 and Km3allotypes.

Antibodies of use may bind to any disease-associated antigen known inthe art. Where the disease state is cancer, for example, many antigensexpressed by or otherwise associated with tumor cells are known in theart, including but not limited to, carbonic anhydrase IX,alpha-fetoprotein (AFP), α-actinin-4, A3, antigen specific for A33antibody, ART-4, B7, Ba 733, BAGE, BrE3-antigen, CA125, CAMEL, CAP-1,CASP-8/m, CCL19, CCL21, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14,CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b,CD33, CD37, CD38, CD40, CD40L, CD44, CD45, CD46, CD52, CD54, CD55, CD59,CD64, CD66a-e, CD67, CD70, CD70L, CD74, CD79a, CD80, CD83, CD95, CD126,CD132, CD133, CD138, CD147, CD154, CDC27, CDK-4/m, CDKN2A, CTLA-4,CXCR4, CXCR7, CXCL12, HIF-1α, colon-specific antigen-p (CSAp), CEA(CEACAM5), CEACAM6, c-Met, DAM, EGFR, EGFRvIII, EGP-1 (Trop-2), EGP-2,ELF2-M, Ep-CAM, fibroblast growth factor (FGF), Flt-1, Flt-3, folatereceptor, G250 antigen, GAGE, gp100, GRO-β, HLA-DR, HM1.24, humanchorionic gonadotropin (HCG) and its subunits, HER2/neu, HMGB-1, hypoxiainducible factor (HIF-1), HSP70-2M, HST-2, Ia, IGF-1R, IFN-γ, IFN-α,IFN-β, IFN-λ, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-2, IL-6,IL-8, IL-12, IL-15, IL-17, IL-18, IL-23, IL-25, insulin-like growthfactor-1 (IGF-1), IGF-1R, KS1-4, Le-Y, LDR/FUT, macrophage migrationinhibitory factor (MIF), MAGE, MAGE-3, MART-1, MART-2, NY-ESO-1, TRAG-3,mCRP, MCP-1, MIP-1A, MIP-1B, MIF, MUC1, MUC2, MUC3, MUC4, MUC5ac, MUC13,MUC16, MUM-1/2, MUM-3, NCA66, NCA95, NCA90, pancreatic cancer mucin,PD-1 receptor, PD-L1 receptor, placental growth factor, p53, PLAGL2,prostatic acid phosphatase, PSA, PRAME, PSMA, PlGF, ILGF, ILGF-1R, IL-6,IL-25, RSS, RANTES, T101, SAGE, S100, survivin, survivin-2B, TAC,TAG-72, tenascin, TRAIL receptors, TNF-α, Tn antigen,Thomson-Friedenreich antigens, tumor necrosis antigens, VEGFR, ED-Bfibronectin, WT-1, 17-1A-antigen, complement factors C3, C3a, C3b, C5a,C5, an angiogenesis marker, bcl-2, bcl-6, Kras, an oncogene marker andan oncogene product (see, e.g., Sensi et al., Clin Cancer Res 2006,12:5023-32; Parmiani et al., J Immunol 2007, 178:1975-79; Novellino etal. Cancer Immunol Immunother 2005, 54:187-207). Preferably, theantibody binds to CEACAM5, CEACAM6, EGP-1 (Trop-2), MUC-16, AFP,MUC5a,c, CD74, CD19, CD20, CD22 or HLA-DR. More preferably, the antibodybinds to Trop-2 or HLA-DR.

Exemplary antibodies that may be utilized include, but are not limitedto, hR1 (anti-IGF-1R, U.S. patent application Ser. No. 13/688,812, filedNov. 29, 2012), hPAM4 (anti-mucin, U.S. Pat. No. 7,282,567), hA20(anti-CD20, U.S. Pat. No. 7,151,164), hA19 (anti-CD19, U.S. Pat. No.7,109,304), hIMMU31 (anti-AFP, U.S. Pat. No. 7,300,655), hLL1(anti-CD74, U.S. Pat. No. 7,312,318), hLL2 (anti-CD22, U.S. Pat. No.5,789,554), hMu-9 (anti-CSAp, U.S. Pat. No. 7,387,772), hL243(anti-HLA-DR, U.S. Pat. No. 7,612,180), hMN-14 (anti-CEACAM5, U.S. Pat.No. 6,676,924), hMN-15 (anti-CEACAM6, U.S. Pat. No. 8,287,865), hRS7(anti-EGP-1, U.S. Pat. No. 7,238,785), hMN-3 (anti-CEACAM6, U.S. Pat.No. 7,541,440), Ab124 and Ab125 (anti-CXCR4, U.S. Pat. No. 7,138,496),the Examples section of each cited patent or application incorporatedherein by reference. More preferably, the antibody is IMMU-31(anti-AFP), hRS7 (anti-Trop-2), hMN-14 (anti-CEACAM5), hMN-3(anti-CEACAM6), hMN-15 (anti-CEACAM6), hLL1 (anti-CD74), hLL2(anti-CD22), hL243 or IMMU-114 (anti-HLA-DR), hA19 (anti-CD19) or hA20(anti-CD20). As used herein, the terms epratuzumab and hLL2 areinterchangeable, as are the terms veltuzumab and hA20, hL243g4P,hL243gamma4P and IMMU-114. In a most preferred embodiment, the antibodyis an anti-Trop-2 antibody, such as hRS7, or an anti-HLA-DR antibody,such as hL243.

Alternative antibodies of use include, but are not limited to, abciximab(anti-glycoprotein IIb/IIIa), alemtuzumab (anti-CD52), bevacizumab(anti-VEGF), cetuximab (anti-EGFR), gemtuzumab (anti-CD33), ibritumomab(anti-CD20), panitumumab (anti-EGFR), rituximab (anti-CD20), tositumomab(anti-CD20), trastuzumab (anti-ErbB2), lambrolizumab (anti-PD-1receptor), atezolizumab (anti-PD-L1), MEDI4736 (anti-PD-L1), nivolumab(anti-PD-1 receptor), ipilimumab (anti-CTLA-4), abagovomab(anti-CA-125), adecatumumab (anti-EpCAM), atlizumab (anti-IL-6receptor), benralizumab (anti-CD125), obinutuzumab (GA101, anti-CD20),CC49 (anti-TAG-72), AB-PG1-XG1-026 (anti-PSMA, U.S. patent applicationSer. No. 11/983,372, deposited as ATCC PTA-4405 and PTA-4406), D2/B(anti-PSMA, WO 2009/130575), tocilizumab (anti-IL-6 receptor),basiliximab (anti-CD25), daclizumab (anti-CD25), efalizumab(anti-CD11a), GA101 (anti-CD20; Glycart Roche), muromonab-CD3 (anti-CD3receptor), natalizumab (anti-α4 integrin), omalizumab (anti-IgE);anti-TNF-α antibodies such as CDP571 (Ofei et al., 2011, Diabetes45:881-85), MTNFAI, M2TNFAI, M3TNFAI, M3TNFABI, M302B, M303 (ThermoScientific, Rockford, Ill.), infliximab (Centocor, Malvern, Pa.),certolizumab pegol (UCB, Brussels, Belgium), anti-CD40L (UCB, Brussels,Belgium), adalimumab (Abbott, Abbott Park, Ill.), Benlysta (Human GenomeSciences); antibodies for therapy of Alzheimer's disease such as Alz 50(Ksiezak-Reding et al., 1987, J Biol Chem 263:7943-47), gantenerumab,solanezumab and infliximab; anti-fibrin antibodies like 59D8, T2G1s,MH1; anti-CD38 antibodies such as MOR03087 (MorphoSys AG), MOR202(Celgene), HuMax-CD38 (Genmab) or daratumumab (Johnson & Johnson);(anti-HIV antibodies such as P4/D10 (U.S. Pat. No. 8,333,971), Ab 75, Ab76, Ab 77 (Paulik et al., 1999, Biochem Pharmacol 58:1781-90), as wellas the anti-HIV antibodies described and sold by Polymun (Vienna,Austria), also described in U.S. Pat. Nos. 5,831,034, 5,911,989, andVcelar et al., AIDS 2007; 21(16):2161-2170 and Joos et al., Antimicrob.Agents Chemother. 2006; 50(5):1773-9, all incorporated herein byreference.

In a preferred embodiment, a drug moiety conjugated to a subjectantibody is selected from camptothecin (CPT) and its analogs andderivatives and is more preferably SN-38. However, other drug moietiesthat may be utilized include taxanes (e.g., baccatin III, taxol),auristatins (e.g., MMAE), calicheamicins, epothilones, anthracyclines(e.g., doxorubicin (DOX), epirubicin, morpholinodoxorubicin(morpholino-DOX), cyanomorpholino-doxorubicin (cyanomorpholino-DOX),2-pyrrolinodoxorubicin (2-PDOX), a prodrug form of 2-PDOX (pro-2-PDOX),topotecan, etoposide, cisplatinum, oxaliplatin or carboplatin; see,e.g., Priebe W (ed.), ACS symposium series 574, published by AmericanChemical Society, Washington D.C., 1995 (332pp) and Nagy et al., Proc.Natl. Acad. Sci. USA 93:2464-2469, 1996). Alternatively, the drug may bea Bruton kinase inhibitor, such as ibrutinib (PCI-32765), PCI-45292,CC-292 (AVL-292), ONO-4059, GDC-0834, LFM-A13 or RN486, or a PI3Kinhibitor, such as idelalisib, Wortmannin, demethoxyviridin, perifosine,PX-866, IPI-145 (duvelisib), BAY 80-6946, BEZ235, RP6530, TGR1202,SF1126, INK1117, GDC-0941, BKM120, XL147, XL765, Palomid 529,GSK1059615, ZSTK474, PWT33597, IC87114, TG100-115, CAL263, PI-103,GNE477, CUDC-907, AEZS-136 or LY294002. Preferably, the antibody orfragment thereof links to at least one chemotherapeutic moiety;preferably 1 to about 5 drug moieties; more preferably 6 to 8 drugmoieties, most preferably about 6 to about 12 drug moieties.

An example of a water soluble CPT derivative is CPT-11. Extensiveclinical data are available concerning CPT-11's pharmacology and its invivo conversion to the active SN-38 (Iyer and Ratain, Cancer ChemotherPharmacol. 42:S31-43 (1998); Mathijssen et al., Clin Cancer Res.7:2182-2194 (2002); Rivory, Ann NY Acad Sci. 922:205-215, 2000)). Theactive form SN-38 is about 2 to 3 orders of magnitude more potent thanCPT-11. In specific preferred embodiments, the immunoconjugate may be anhMN-14-SN-38, hMN-3-SN-38, hMN-15-SN-38, IMMU-31-SN-38, hRS7-SN-38,hA20-SN-38, hL243-SN-38, hLL1-SN-38 or hLL2-SN-38 conjugate.

Various embodiments may concern use of the subject methods andcompositions to treat a cancer, including but not limited tonon-Hodgkin's lymphomas, B-cell acute and chronic lymphoid leukemias,Burkitt lymphoma, Hodgkin's lymphoma, acute large B-cell lymphoma, hairycell leukemia, acute myeloid leukemia, chronic myeloid leukemia, acutelymphocytic leukemia, chronic lymphocytic leukemia, T-cell lymphomas andleukemias, multiple myeloma, Waldenstrom's macroglobulinemia,carcinomas, melanomas, sarcomas, gliomas, bone, and skin cancers. Thecarcinomas may include carcinomas of the oral cavity, esophagus,gastrointestinal tract, pulmonary tract, lung, stomach, colon, rectum,breast, ovary, prostate, uterus, endometrium, cervix, urinary bladder,pancreas, bone, brain, connective tissue, thyroid, liver, gall bladder,urinary bladder (urothelial), kidney, skin, central nervous system andtestes.

In certain embodiments involving treatment of cancer, the antibodies orimmunoconjugates may be used in combination with surgery, radiationtherapy, chemotherapy, immunotherapy with naked antibodies, includingcheckpoint-inhibiting antibodies, radioimmunotherapy, immunomodulators,vaccines, and the like. Most preferably, the antibody or immunoconjugateis used in combination with a PARP inhibitor, microtubule inhibitor,Bruton kinase inhibitor and/or PI3K inhibitor. These combinationtherapies can allow lower doses of each therapeutic to be given in suchcombinations, thus reducing certain severe side effects, and potentiallyreducing the courses of therapy required. When there is no or minimaloverlapping toxicity, full doses of each can also be given.

Preferred optimal dosing of immunoconjugates may include a dosage ofbetween 1 mg/kg and 20 mg/kg, more preferably 4 to 16 mg/kg, mostpreferably 8 to 10 mg/kg, preferably given either weekly, twice weekly,every other week, or every third week. The optimal dosing schedule mayinclude treatment cycles of two consecutive weeks of therapy followed byone, two, three or four weeks of rest, or alternating weeks of therapyand rest, or one week of therapy followed by two, three or four weeks ofrest, or three weeks of therapy followed by one, two, three or fourweeks of rest, or four weeks of therapy followed by one, two, three orfour weeks of rest, or five weeks of therapy followed by one, two,three, four or five weeks of rest, or administration once every twoweeks, once every three weeks or once a month. Treatment may be extendedfor any number of cycles, preferably at least 2, at least 4, at least 6,at least 8, at least 10, at least 12, at least 14, or at least 16cycles. Exemplary dosages of use may include 1 mg/kg, 2 mg/kg, 3 mg/kg,4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, 11mg/kg, 12 mg/kg, 13 mg/kg, 14 mg/kg, 15 mg/kg, 16 mg/kg, 17 mg/kg, and18 mg/kg. The person of ordinary skill will realize that a variety offactors, such as age, general health, specific organ function or weight,as well as effects of prior therapy on specific organ systems (e.g.,bone marrow) may be considered in selecting an optimal dosage ofimmunoconjugate, and that the dosage and/or frequency of administrationmay be increased or decreased during the course of therapy. The dosagemay be repeated as needed, with evidence of tumor shrinkage observedafter as few as 4 to 8 doses. The optimized dosages and schedules ofadministration disclosed herein show unexpected superior efficacy andreduced toxicity in human subjects, which could not have been predictedfrom animal model studies. Surprisingly, the superior efficacy allowstreatment of tumors that were previously found to be resistant to one ormore standard anti-cancer therapies, including the parental compound,CPT-11, from which SN-38 is derived in vivo.

The subject methods may include use of CT and/or PET/CT, or MRI, tomeasure tumor response at regular intervals. Blood levels of tumormarkers, such as CEA (carcinoembryonic antigen), CA19-9, AFP, CA 15.3,or PSA, may also be monitored. Dosages and/or administration schedulesmay be adjusted as needed, according to the results of imaging and/ormarker blood levels.

A surprising result with the instant claimed compositions and methods isthe unexpected tolerability of high doses of antibody-drug conjugatewhen given to patients, even with repeated infusions, with onlyrelatively low-grade toxicities of nausea and vomiting observed, ormanageable neutropenia. Preferably, the incidence of grade 3 or higherside effects, such as anemia, diarrhea, nausea, vomiting or neutropenia,is limited to 26% or less of the treated population. More preferably,the incidence of grade 3 or higher diarrhea and neutropenia occurs in26% or less of the treated population. A further surprising result isthe lack of accumulation of the antibody-drug conjugate, unlike otherproducts that have conjugated SN-38 to albumin, PEG or other carriers,to nontarget tissues or target tissues where the MAb is not accessible.The lack of accumulation is associated with improved tolerability andlack of serious toxicity even after repeated or increased dosing. Thesesurprising results allow optimization of dosage and delivery schedule,with unexpectedly high efficacies and low toxicities; i.e., a highertherapeutic index than when the parental drug to SN-38, irinotecan, isgiven alone or in combination with other drugs. This is underscored bythe black box warning on the product labeling of irinotecan, required byFDA, stating the high severe early and late diarrhea resulting fromtherapy with irinotecan. This is not experienced to the same extent andfrequency with these SN-38-containing ADCs. Even febrile neutropenia ismuch lower with the SN-38-ADCs described herein than with irinotecantherapy.

The claimed methods provide for shrinkage of solid tumors, inindividuals with previously resistant cancers, of 15% or more,preferably 20% or more, preferably 30% or more, more preferably 40% ormore in size (as measured by summing the longest diameter of targetlesions, as per RECIST or RECIST 1.1). The person of ordinary skill willrealize that tumor size may be measured by a variety of differenttechniques, such as total tumor volume, maximal tumor size in anydimension or a combination of size measurements in several dimensions.This may be with standard radiological procedures, such as computedtomography, magnet resonance imaging, ultrasonography, and/orpositron-emission tomography. The means of measuring size is lessimportant than observing a trend of decreasing tumor size with antibodyor immunoconjugate treatment, preferably resulting in elimination of thetumor. However, to comply with RECIST guidelines, CT or MRI is preferredon a serial basis, and should be repeated to confirm measurements.

While the antibody or immunoconjugate may be administered as a periodicbolus injection, in alternative embodiments the antibody orimmunoconjugate may be administered by continuous infusion. In order toincrease the Cmax and extend the PK of the antibody or immunoconjugatein the blood, a continuous infusion may be administered for example byindwelling catheter. Such devices are known in the art, such asHICKMAN®, BROVIAC® or PORT-A-CATH® catheters (see, e.g., Skolnik et al.,Ther Drug Monit 32:741-48, 2010) and any such known indwelling cathetermay be used. A variety of continuous infusion pumps are also known inthe art and any such known infusion pump may be used. The dosage rangefor continuous infusion may be between 0.1 and 3.0 mg/kg per day. Morepreferably, these immunoconjugates can be administered by intravenousinfusions over relatively short periods of 2 to 5 hours, more preferably2-3 hours.

In particularly preferred embodiments, the antibodies orimmunoconjugates and dosing schedules may be efficacious in patientsresistant to standard therapies. For example, an hRS7-SN-38immunoconjugate may be administered to a patient who has not respondedto prior therapy with irinotecan, the parent agent of SN-38.Surprisingly, the irinotecan-resistant patient may show a partial oreven a complete response to hRS7-SN-38. The ability of theimmunoconjugate to specifically target the tumor tissue may overcometumor resistance by improved targeting and enhanced delivery of thetherapeutic agent. A specific preferred subject may be a patient with aTrop-2-positive breast, ovarian, cervical, endometrial, lung, prostate,colon, rectum, stomach, esophageal, bladder (urothelial), renal,pancreatic, brain, thyroid, epithelial or head-and-neck cancer.Preferably, the cancer is metastatic cancer. More preferably, thepatient has previously failed treatment with at least one standardanti-cancer therapy. Most preferably, the patient has previously failedtherapy with irinotecan (CPT-11), the parent compound of SN-38. Inalternative preferred embodiments, the cancer is TNBC, non-TNBC,endometrial, lung, or ovarian cancer.

In certain preferred embodiments, an antibody or immunoconjugate, suchas sacituzumab govitecan, may be used in combination therapy with atleast one microtubule inhibitor. A number of microtubule inhibitors areknown in the art, such as vinca alkaloids (e.g., vincristine,vinblastine), taxanes (e.g., paclitaxel), maytansinoids (e.g.,mertansine) and auristatins. Other known microtubule inhibitors includedemecolcine, nocodazole, epothilone, docetaxel, discodermolide,colchicine, combrestatin, podophyllotoxin, CI-980, phenylahistins,steganacins, curacins, 2-methoxy estradiol, E7010, methoxybenzenesuflonamides, vinorelbine, vinflunine, vindesine, dolastatins,spongistatin, rhizoxin, tasidotin, halichondrins, hemiasterlins,cryptophycin 52, MMAE and eribulin mesylate (see, e.g., Dumontet &Jordan, 2010, Nat Rev Drug Discov 9:790-803). Any such known microtubuleinhibitor may be used in combination with an antibody or antibody-drugconjugate (ADC). Preferably, the microtubule inhibitor is one thatexhibits synergistic effects when used in combination with an antibodyor ADC. One potent example is SN-38-conjugated antibody, such assacituzumab govitecan or labetuzumab govitecan (targeting CEACAM5)expressed by many solid cancers. Most preferably, the microtubuleinhibitor is paclitaxel or eribulin mesylate.

In other preferred embodiments, the antibody or ADC may be used incombination therapy with at least one PARP inhibitor. One such ADC iscomprised of SN-38 conjugated to a tumor-targeting antibody, such assacituzumab govitecan or labetuzumab govitecan. A number of PARPinhibitors are known in the art, such as olaparib, talazoparib(BMN-673), rucaparib, veliparib, niraparib, iniparib, CEP 9722, MK 4827,BGB-290, ABT-888, AG014699, BSI-201, CEP-8983 and 3-aminobenzamide (see,e.g., Rouleau et al., 2010, Nat Rev Cancer 10:293-301, Bao et al., 2015,Oncotarget [Epub ahead of print, Sep. 22, 2015]). Any such known PARPinhibitor may be used in combination with an antibody or ADC, such as,for example, an SN-38-antibody conjugate or a P2PDox-antibody conjugate.Preferably, the PARP inhibitor is one that exhibits synergistic effectswhen used in combination with the antibody or ADC. This has beenvalidated when using an SN-38-conjugated antibody, such as sacituzumabgovitecan. Most preferably, the PARP inhibitor is olaparib or rucaparib.

In still other embodiments, an antibody or immunoconjugate may be usedin combination with a Bruton kinase inhibitor or PI3K inhibitor.Exemplary Bruton kinase inhibitors include, but are not limited to,ibrutinib (PCI-32765), PCI-45292, CC-292 (AVL-292), ONO-4059, GDC-0834,LFM-A13 or RN486. Exemplary PI3K inhibitors include, but are not limitedto, idelalisib, Wortmannin, demethoxyviridin, perifosine, PX-866,IPI-145 (duvelisib), BAY 80-6946, BEZ235, RP6530, TGR1202, SF1126,INK1117, GDC-0941, BKM120, XL147, XL765, Palomid 529, GSK1059615,ZSTK474, PWT33597, IC_(87114,) TG100-115, CAL263, PI-103, GNE477,CUDC-907, AEZS-136 or LY294002. Any Bruton kinase or PI3K inhibitorsknown in the art may be utilized in the claimed combination therapy.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A. Synergy of IMMU-132 plus Olaparib in BRCA1/2 w.t. TNBC(MDA-MB-468). Dose/response curves for each agent alone were firsttested to determine single agent IC₁₀, IC₂₀, or IC₃₀-values after a 96-hincubation. In combination assays, one agent (e.g., IMMU-132) was testedon a given cell line across a range of concentrations (i.e.,dose/response curves). One set of wells only received IMMU-132. Anotherset received IMMU-132 as dose/response with a constant amount ofolaparib (e.g., IC₁₀-concentration). Two other sets used olaparib atIC₂₀ or IC₃₀ concentrations. For each IMMU-132 dose/response curve, theIC₅₀-value was determined from these data. FIG. 1A shows changes inIC₅₀-values of olaparib when combined with constant amounts of IMMU-132(0.6, 1.1 or 1.8 nM SN-38 equivalents).

FIG. 1B. Synergy of IMMU-132 plus Olaparib in BRCA1/2 w.t. TNBC(MDA-MB-468). Dose/response curves for each agent alone were firsttested to determine single agent IC₁₀, IC₂₀, or IC₃₀-values after a 96-hincubation. In combination assays, one agent (e.g., IMMU-132) was testedon a given cell line across a range of concentrations (i.e.,dose/response curves). One set of wells only received IMMU-132. Anotherset received IMMU-132 as dose/response with a constant amount ofolaparib (e.g., IC₁₀-concentration). Two other sets used olaparib atIC₂₀ or IC₃₀ concentrations. For each IMMU-132 dose/response curve, theIC₅₀-value was determined from these data. FIG. 1B shows the same cellline showing changes in IMMU-132 IC₅₀-values when combined with constantamounts of olaparib (1, 2, or 3 mM).

FIG. 1C. Synergy of IMMU-132 plus Olaparib in BRCA1/2 w.t. TNBC(MDA-MB-468). Dose/response curves for each agent alone were firsttested to determine single agent IC₁₀, IC₂₀, or IC₃₀-values after a 96-hincubation. In combination assays, one agent (e.g., IMMU-132) was testedon a given cell line across a range of concentrations (i.e.,dose/response curves). One set of wells only received IMMU-132. Anotherset received IMMU-132 as dose/response with a constant amount ofolaparib (e.g., IC₁₀-concentration). Two other sets used olaparib atIC₂₀ or IC₃₀ concentrations. For each IMMU-132 dose/response curve, theIC₅₀-value was determined from these data. FIG. 1C shows an isobologramof normalized IC₅₀-values from three separate experiments testing theinteraction of IMMU-132 and olaparib, clearly showing a synergisticinteraction.

FIG. 2A. Tumor growth inhibition of combined IMMU-132 and Olaparib inTNBC: BRCA1/2 and PTEN defective tumors. Tumor-bearing mice (TV˜0.3 cm³)were treated with Olaparib (1 mg; ˜50 mg/kg, i.p. on a M-F schedule; redarrows) or IMMU-132 (i.v. weekly, black arrows). A non-tumor-targetinganti-CD20 SN-38-ADC was used as a control. HCC1806 is aBRCA1/2-defective TNBC tumor line. Olaparib alone had no significantanti-tumor effects. IMMU-132 alone significantly inhibited tumor growthcompared to all control groups (P<0.0106, AUC). IMMU-132 plus olaparibfurther improved anti-tumor responses significantly compared to allgroups (P<0.0019; AUC). Mice in the combination group have yet to reachmedian survival (>80.5 days) which is more than 2- and 4-fold longerthan IMMU-132 or olaparib monotherapy, respectively (P<0.0083).

FIG. 2B. Tumor growth inhibition of combined IMMU-132 and Olaparib inTNBC: BRCA1/2 and PTEN defective tumors. Tumor-bearing mice (TV˜0.3 cm³)were treated with Olaparib (1 mg; ˜50 mg/kg, i.p. on a M-F schedule;light arrows) or IMMU-132 (i.v. weekly, dark arrows). Anon-tumor-targeting anti-CD20 SN-38-ADC was used as a control. In theBRCA1/2 w.t., PTEN-defective MDA-MB-468 tumors, IMMU-132 alone hadsignificant anti-tumor effects compared to all control groups (P<0.0098;AUC). However, the combination of IMMU-132 plus olaparib inhibited tumorgrowth significantly better than either IMMU-132 or olaparib alone(P=0.004; AUC). This translates into a significant survival benefit whencompared to all other groups (P<0.045).

FIG. 3A. Tumor growth inhibition of combined IMMU-132 and microtubuleinhibitors in human TNBC tumor xenografts. Tumor-bearing mice (TV˜0.3cm³) were treated with either paclitaxel or eribulin mesylate, asindicated, either alone or with IMMU-132. Therapy was administered asweekly injections for two weeks with one week off before repeating. Anon-tumor-targeting anti-CD20 SN-38-ADC was used as a control ADC.HCC1806 tumor-bearing mice treated with the combination of IMMU-132 pluspaclitaxel significantly inhibited tumor growth when compared toIMMU-132 alone (P<0.0195, AUC).

FIG. 3B. Tumor growth inhibition of combined IMMU-132 and microtubuleinhibitors in human TNBC tumor xenografts. Tumor-bearing mice (TV˜0.3cm³) were treated with either paclitaxel or eribulin mesylate, asindicated, either alone or with IMMU-132. Therapy was administered asweekly injections for two weeks with one week off before repeating. Anon-tumor-targeting anti-CD20 SN-38-ADC was used as a control ADC.HCC1806 tumor-bearing mice treated with the combination of IMMU-132 pluspaclitaxel showed a significant survival benefit when compared to allother treatments (P<0.006, log-rank).

FIG. 3C. Tumor growth inhibition of combined IMMU-132 and microtubuleinhibitors in human TNBC tumor xenografts. Mice bearing MDA-MB-468tumors demonstrated significant anti-tumor effects when IMMU-132 (100 or200 mg) was combined with paclitaxel when compared to mice receivingmonotherapy (P<0.0328, AUC).

FIG. 3D. Tumor growth inhibition of combined IMMU-132 and microtubuleinhibitors in human TNBC tumor xenografts. Eribulin mesylate alonesignificantly inhibits MDA-MB-468 tumor growth (P=0.0019, AUC).Importantly, the combination of IMMU-132 plus eribulin mesylate resultedin significant tumor regressions with mean tumor volumes regressing to0.026±0.033 cm³ versus 0.177±0.087 cm³ and 0.344±302 cm³ for IMMU-132and eribulin monotherapy groups, respectively (P<0.0009 combo vs. allother therapy groups, AUC).

FIG. 3E. Tumor growth inhibition of combined IMMU-132 and microtubuleinhibitors in human TNBC tumor xenografts. IMMU-132 plus eribulinmesylate produced significant anti-tumor effects when compared to allother treatment groups (P<0.0432; paired, two-tailed t-Test).

FIG. 3F. Tumor growth inhibition of combined IMMU-132 and microtubuleinhibitors in human TNBC tumor xenografts. IMMU-132 plus eribulinmesylate provided a significant survival benefit compared to all groups,except the combination of eribulin mesylate plus control ADC (P<0.0284).

FIG. 4. Synergistic activity of IMMU-132 plus Olaparib in various humanTNBC cell lines. Summary of calculated combination index (CI)-values forvarious cell lines shown. CI numbers are calculated using the followingformula: CI=Da/Dxa+Db/Dxb, where

-   -   Da=IC₅₀-dose of IMMU-132 when used in combination with a        constant amount of Olaparib    -   Db=IC₅₀-dose of Olaparib when used in combination with a        constant amount of IMMU-132    -   Dxa=IC₅₀-dose of IMMU-132 when used alone    -   Dxb=IC₅₀-dose of Olaparib when used alone    -   ^(a)IMMU-132 IC₅₀-value (nM) when combined with olaparib.    -   ^(b)Olaparib IC₅₀-value (nM) when combined with IMMU-132.    -   ^(c)CI combination index number (CI<1.0 is indicative of        synergy).

FIG. 5A. Effect of IMMU-114 on IC₅₀ of ibrutinib.

FIG. 5B. Effect of IMMU-114 on IC₅₀ of idelalisib.

FIG. 6A. Isobologram demonstrating an additive effect of combinedIMMU-114 with a Bruton kinase inhibitor (ibrutinib) in a human CLL cellline. Human chronic B-cell leukemia cells (JVM-3) were incubated in96-well plates with a Bruton kinase inhibitor (ibrutinib) at dosesranging from 1×10⁻⁵ to 3.9×10⁻⁹M. One set of cells received only theinhibitor while other sets received the inhibitor plus a constant amountof IMMU-114 (0.25, 0.5, 0.75 or 1 nM). Plates were incubated for 96 h.Cell viability was assessed by MTS assay and dose/response curves weregenerated for each condition. IC₅₀-values were determined using PrismGraph-Pad. Data were normalized and isobologram generated, indicating anadditive effect for ibrutinib when combined with IMMU-114

FIG. 6B. Isobologram demonstrating an additive effect of combinedIMMU-114 with a PI3K inhibitor (idelalisib) in a human CLL cell line.The studies were performed as described in the legend to FIG. 6A. Theisobologram indicated an additive effect for idelalisib when combinedwith IMMU-114.

FIG. 7. In vivo therapy of athymic nude mice, bearing Capan 1 humanpancreatic carcinoma, with MAb-CL2A-SN-38 conjugates.

FIG. 8. In vivo therapy of athymic nude mice, bearing BxPC3 humanpancreatic carcinoma, with MAb-CL2A-SN-38 conjugates.

FIG. 9. In vivo therapy of athymic nude mice, bearing LS174T human coloncarcinoma, with hMN-14-CL2A-SN-38 conjugate.

FIG. 10. Survival curves of hMN14-CL-SN-38 treated mice bearing GW-39lung metastatic disease.

FIG. 11A. Therapeutic efficacy of hRS7-SN-38 ADC in mice bearing humannon-small cell lung tumor xenografts. Mice bearing Calu-3 tumors (N=5-7)were injected with hRS7-CL2-SN-38 every 4 days for a total of 4injections (q4dx4). All the ADCs and controls were administered in theamounts indicated (expressed as amount of SN-38 per dose; longarrows=conjugate injections, short arrows=irinotecan injections).

FIG. 11B. Therapeutic efficacy of hRS7-SN-38 ADC in mice bearing humancolorectal tumor xenografts. COLO 205 tumor-bearing mice (N=5) wereinjected 8 times (q4dx8) with the ADC or every 2 days for a total of 5injections (q2dx5) with the MTD of irinotecan. All the ADCs and controlswere administered in the amounts indicated (expressed as amount of SN-38per dose; long arrows=conjugate injections, short arrows=irinotecaninjections).

FIG. 11C. Therapeutic efficacy of hRS7-SN-38 ADC in mice bearing humanpancreatic cancer xenografts. Capan-1 (N=10) tumor-bearing mice (N=10)were treated twice weekly for 4 weeks with the agents indicated. All theADCs and controls were administered in the amounts indicated (expressedas amount of SN-38 per dose; long arrows=conjugate injections, shortarrows=irinotecan injections).

FIG. 11D. Therapeutic efficacy of hRS7-SN-38 ADC in mice bearing humanpancreatic cancer xenografts. BxPC-3 tumor-bearing mice (N=10) weretreated twice weekly for 4 weeks with the agents indicated. All the ADCsand controls were administered in the amounts indicated (expressed asamount of SN-38 per dose; long arrows=conjugate injections, shortarrows=irinotecan injections).

FIG. 11E. Therapeutic efficacy of hRS7-SN-38 ADC in mice bearing humansquamous cell lung carcinoma xenografts. In addition to ADC given twiceweekly for 4 week, SK-MES-1 tumor-bearing (N=8) mice received the MTD ofCPT-11 (q2dx5). All the ADCs and controls were administered in theamounts indicated (expressed as amount of SN-38 per dose; longarrows=conjugate injections, short arrows=irinotecan injections).

FIG. 12A. Comparative efficacy of epratuzumab (Emab)-SN-38 andveltuzumab (Vmab)-SN-38 conjugates in the subcutaneous Ramos model. Nudemice (N=10 per group) with tumors averaging approximately 0.35 cm³(0.20-0.55 cm³) were administered 0.25 mg of Emab-SN-38 twice weekly for4 weeks.

FIG. 12B. Comparative efficacy of epratuzumab (Emab)-SN-38 andveltuzumab (Vmab)-SN-38 conjugates in the subcutaneous Ramos model. Nudemice (N=10 per group) with tumors averaging approximately 0.35 cm³(0.20-0.55 cm³) were administered 0.25 mg of Vmab-SN-38 twice weekly for4 weeks.

FIG. 12C. Comparative efficacy of epratuzumab (Emab)-SN-38 andveltuzumab (Vmab)-SN-38 conjugates in the subcutaneous Ramos model. Nudemice (N=10 per group) with tumors averaging approximately 0.35 cm³(0.20-0.55 cm³) were administered 0.5 mg of Emab-SN-38 twice weekly for4 weeks.

FIG. 12D. Comparative efficacy of epratuzumab (Emab)-SN-38 andveltuzumab (Vmab)-SN-38 conjugates in the subcutaneous Ramos model. Nudemice (N=10 per group) with tumors averaging approximately 0.35 cm³(0.20-0.55 cm³) were administered 0.5 mg of Vmab-SN-38 twice weekly for4 weeks.

FIG. 13A. Specificity of Emab anti-CD22-SN-38 conjugate (solid line)versus an irrelevant labetuzumab (Lmab)-SN-38 conjugate (dashed line) innude mice bearing subcutaneous Ramos tumors. Animals were given twiceweekly doses of 75 μg of each conjugate per dose (54.5 μg/kg of SN-38,based on average weight of 22 g) intraperitoneally for 4 weeks. Survivalbased on time-to-progression (TTP) to 3.0 cm³, with tumors starting atan average size of 0.4 cm³. P values comparing median survival (shown)for Emab-SN-38 to Lmab-SN-38 conjugate are shown in each panel. C,survival curves (solid gray) for another group of animals given weeklyintraperitoneal injections of irinotecan (6.5 μg/dose; SN-38 equivalentsapproximately the same as the 250-μg dose of the Emab-SN-38 conjugate).

FIG. 13B. Specificity of Emab anti-CD22-SN-38 conjugate (solid line)versus an irrelevant labetuzumab (Lmab)-SN-38 conjugate (dashed line) innude mice bearing subcutaneous Ramos tumors. Animals were given twiceweekly doses of 125 μg of each conjugate per dose (91 μg/kg of SN-38,based on average weight of 22 g) intraperitoneally for 4 weeks. Survivalbased on time-to-progression (TTP) to 3.0 cm³, with tumors starting atan average size of 0.4 cm³. P values comparing median survival (shown)for Emab-SN-38 to Lmab-SN-38 conjugate are shown in each panel. C,survival curves (solid gray) for another group of animals given weeklyintraperitoneal injections of irinotecan (6.5 μg/dose; SN-38 equivalentsapproximately the same as the 250-μg dose of the Emab-SN-38 conjugate).

FIG. 13C. Specificity of Emab anti-CD22-SN-38 conjugate (solid line)versus an irrelevant labetuzumab (Lmab)-SN-38 conjugate (dashed line) innude mice bearing subcutaneous Ramos tumors. Animals were given twiceweekly doses of 250 μg of each conjugate per dose (182 μg/kg of SN-38,based on average weight of 22 g) intraperitoneally for 4 weeks. Survivalbased on time-to-progression (TTP) to 3.0 cm³, with tumors starting atan average size of 0.4 cm³. P values comparing median survival (shown)for Emab-SN-38 to Lmab-SN-38 conjugate are shown in each panel. Survivalcurves (solid gray) are also shown for another group of animals givenweekly intraperitoneal injections of irinotecan (6.5 μg/dose; SN-38equivalents approximately the same as the 250-μg dose of the Emab-SN-38conjugate).

FIG. 14. History of prior treatment of patient, before administeringIMMU-130 (labetuzumab-SN-38). Prior treatment included stage IV CRCcoloectomy/hepatectomy (partial lobe), radiofrequency ablation therapyof liver metasteses, wedge resection of lung metasteses, andchemotherapy with irinotecan/oxaliplatin, Folfirinox,Folfirinox+bevacizumab, bevacizumab+5-FU/leucovorin, FolFiri,Folfiri+cetuximab, and cetuximab alone. The patient received doses of 16mg/kg of IMMU-130 by slow IV infusion every other week for a total of 17treatment doses.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

In the description that follows, a number of terms are used and thefollowing definitions are provided to facilitate understanding of theclaimed subject matter. Terms that are not expressly defined herein areused in accordance with their plain and ordinary meanings.

Unless otherwise specified, a or an means “one or more.”

The term about is used herein to mean plus or minus ten percent (10%) ofa value. For example, “about 100” refers to any number between 90 and110.

An antibody, as used herein, refers to a full-length (i.e., naturallyoccurring or formed by normal immunoglobulin gene fragmentrecombinatorial processes) immunoglobulin molecule (e.g., an IgGantibody) or an antigen-binding portion of an immunoglobulin molecule,such as an antibody fragment. An antibody or antibody fragment may beconjugated or otherwise derivatized within the scope of the claimedsubject matter. Such antibodies include but are not limited to IgG1,IgG2, IgG3, IgG4 (and IgG4 subforms), as well as IgA isotypes. As usedbelow, the abbreviation “MAb” may be used interchangeably to refer to anantibody, antibody fragment, monoclonal antibody or multispecificantibody.

An antibody fragment is a portion of an antibody such as F(ab′)₂,F(ab)₂, Fab′, Fab, Fv, scFv (single chain Fv), single domain antibodies(DABs or VHHs) and the like, including the half-molecules of IgG4 citedabove (van der Neut Kolfschoten et al. (Science 2007; 317(14September):1554-1557). Regardless of structure, an antibody fragment ofuse binds with the same antigen that is recognized by the intactantibody. The term “antibody fragment” also includes synthetic orgenetically engineered proteins that act like an antibody by binding toa specific antigen to form a complex. For example, antibody fragmentsinclude isolated fragments consisting of the variable regions, such asthe “Fv” fragments consisting of the variable regions of the heavy andlight chains and recombinant single chain polypeptide molecules in whichlight and heavy variable regions are connected by a peptide linker(“scFv proteins”). The fragments may be constructed in different ways toyield multivalent and/or multispecific binding forms.

A naked antibody is generally an entire antibody that is not conjugatedto a therapeutic agent. A naked antibody may exhibit therapeutic and/orcytotoxic effects, for example by Fc-dependent functions, such ascomplement fixation (CDC) and ADCC (antibody-dependent cellcytotoxicity). However, other mechanisms, such as apoptosis,anti-angiogenesis, anti-metastatic activity, anti-adhesion activity,inhibition of heterotypic or homotypic adhesion, and interference insignaling pathways, may also provide a therapeutic effect. Nakedantibodies include polyclonal and monoclonal antibodies, naturallyoccurring or recombinant antibodies, such as chimeric, humanized orhuman antibodies and fragments thereof. In some cases a “naked antibody”may also refer to a “naked” antibody fragment. As defined herein,“naked” is synonymous with “unconjugated,” and means not linked orconjugated to a therapeutic agent.

A chimeric antibody is a recombinant protein that contains the variabledomains of both the heavy and light antibody chains, including thecomplementarity determining regions (CDRs) of an antibody derived fromone species, preferably a rodent antibody, more preferably a murineantibody, while the constant domains of the antibody molecule arederived from those of a human antibody. For veterinary applications, theconstant domains of the chimeric antibody may be derived from that ofother species, such as a primate, cat or dog.

A humanized antibody is a recombinant protein in which the CDRs from anantibody from one species; e.g., a murine antibody, are transferred fromthe heavy and light variable chains of the murine antibody into humanheavy and light variable domains (framework regions). The constantdomains of the antibody molecule are derived from those of a humanantibody. In some cases, specific residues of the framework region ofthe humanized antibody, particularly those that are touching or close tothe CDR sequences, may be modified, for example replaced with thecorresponding residues from the original murine, rodent, subhumanprimate, or other antibody.

A human antibody is an antibody obtained, for example, from transgenicmice that have been “engineered” to produce human antibodies in responseto antigenic challenge. In this technique, elements of the human heavyand light chain loci are introduced into strains of mice derived fromembryonic stem cell lines that contain targeted disruptions of theendogenous heavy chain and light chain loci. The transgenic mice cansynthesize human antibodies specific for various antigens, and the micecan be used to produce human antibody-secreting hybridomas. Methods forobtaining human antibodies from transgenic mice are described by Greenet al., Nature Genet. 7:13 (1994), Lonberg et al., Nature 368:856(1994), and Taylor et al., Int. Immun. 6:579 (1994). A fully humanantibody also can be constructed by genetic or chromosomal transfectionmethods, as well as phage display technology, all of which are known inthe art. See for example, McCafferty et al., Nature 348:552-553 (1990)for the production of human antibodies and fragments thereof in vitro,from immunoglobulin variable domain gene repertoires from unimmunizeddonors. In this technique, human antibody variable domain genes arecloned in-frame into either a major or minor coat protein gene of afilamentous bacteriophage, and displayed as functional antibodyfragments on the surface of the phage particle. Because the filamentousparticle contains a single-stranded DNA copy of the phage genome,selections based on the functional properties of the antibody alsoresult in selection of the gene encoding the antibody exhibiting thoseproperties. In this way, the phage mimics some of the properties of theB cell. Phage display can be performed in a variety of formats, fortheir review, see e.g. Johnson and Chiswell, Current Opinion inStructural Biology 3:5564-571 (1993). Human antibodies may also begenerated by in vitro activated B cells. See U.S. Pat. Nos. 5,567,610and 5,229,275, the Examples section of each of which is incorporatedherein by reference.

A therapeutic agent is an atom, molecule, or compound that is useful inthe treatment of a disease. Examples of therapeutic agents include, butare not limited to, antibodies, antibody fragments, immunoconjugates,drugs, cytotoxic agents, pro-apopoptotic agents, toxins, nucleases(including DNAses and RNAses), hormones, immunomodulators, chelators,boron compounds, photoactive agents or dyes, radionuclides,oligonucleotides, interference RNA, siRNA, RNAi, anti-angiogenic agents,chemotherapeutic agents, cyokines, chemokines, prodrugs, enzymes,binding proteins or peptides or combinations thereof.

An immunoconjugate is an antibody, antigen-binding antibody fragment,antibody complex or antibody fusion protein that is conjugated to atleast one therapeutic agent. Conjugation may be covalent ornon-covalent. Preferably, conjugation is covalent.

As used herein, the term antibody fusion protein is arecombinantly-produced antigen-binding molecule in which one or morenatural antibodies, single-chain antibodies or antibody fragments arelinked to another moiety, such as a protein or peptide, a toxin, acytokine, a hormone, etc. In certain preferred embodiments, the fusionprotein may comprise two or more of the same or different antibodies,antibody fragments or single-chain antibodies fused together, which maybind to the same epitope, different epitopes on the same antigen, ordifferent antigens.

An immunomodulator is a therapeutic agent that when present, alters,suppresses or stimulates the body's immune system. Typically, animmunomodulator of use stimulates immune cells to proliferate or becomeactivated in an immune response cascade, such as macrophages, dendriticcells, B-cells, and/or T-cells. However, in some cases animmunomodulator may suppress proliferation or activation of immunecells. An example of an immunomodulator as described herein is acytokine, which is a soluble small protein of approximately 5-20 kDathat is released by one cell population (e.g., primed T-lymphocytes) oncontact with specific antigens, and which acts as an intercellularmediator between cells. As the skilled artisan will understand, examplesof cytokines include lymphokines, monokines, interleukins, and severalrelated signaling molecules, such as tumor necrosis factor (TNF) andinterferons. Chemokines are a subset of cytokines. Certain interleukinsand interferons are examples of cytokines that stimulate T cell or otherimmune cell proliferation. Exemplary interferons include interferon-α,interferon-β, interferon-γ and interferon-λ.

CPT is an abbreviation for camptothecin, and as used in the presentapplication CPT represents camptothecin itself or an analog orderivative of camptothecin, such as SN-38. The structures ofcamptothecin and some of its analogs, with the numbering indicated andthe rings labeled with letters A-E, are given in formula 1 in Chart 1below.

General Antibody Techniques

Techniques for preparing monoclonal antibodies against virtually anytarget antigen are well known in the art. See, for example, Köhler andMilstein, Nature 256: 495 (1975), and Coligan et al. (eds.), CURRENTPROTOCOLS IN IMMUNOLOGY, VOL. 1, pages 2.5.1-2.6.7 (John Wiley & Sons1991). The person of ordinary skill will realize that, where humansubjects are to be treated, the antibodies preferably bind to humanantigens. Briefly, monoclonal antibodies can be obtained by injectingmice with a composition comprising an antigen, removing the spleen toobtain B-lymphocytes, fusing the B-lymphocytes with myeloma cells toproduce hybridomas, cloning the hybridomas, selecting positive cloneswhich produce antibodies to the antigen, culturing the clones thatproduce antibodies to the antigen, and isolating the antibodies from thehybridoma cultures.

MAbs can be isolated and purified from hybridoma cultures by a varietyof well-established techniques. Such isolation techniques includeaffinity chromatography with Protein-A or Protein-G Sepharose,size-exclusion chromatography, and ion-exchange chromatography. See, forexample, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. Also, seeBaines et al., “Purification of Immunoglobulin G (IgG),” in METHODS INMOLECULAR BIOLOGY, VOL. 10, pages 79-104 (The Humana Press, Inc. 1992).

After the initial raising of antibodies to the immunogen, the antibodiescan be sequenced and subsequently prepared by recombinant techniques.Humanization and chimerization of murine antibodies and antibodyfragments are well known to those skilled in the art, as discussedbelow.

The skilled artisan will realize that the claimed methods andcompositions may utilize any of a wide variety of antibodies known inthe art. Antibodies of use may be commercially obtained from a widevariety of known sources. For example, a variety of antibody secretinghybridoma lines are available from the American Type Culture Collection(ATCC, Manassas, Va.). A large number of antibodies against variousdisease targets, including but not limited to tumor-associated antigens,have been deposited at the ATCC and/or have published variable regionsequences and are available for use in the claimed methods andcompositions. See, e.g., U.S. Pat. Nos. 7,312,318; 7,282,567; 7,151,164;7,074,403; 7,060,802; 7,056,509; 7,049,060; 7,045,132; 7,041,803;7,041,802; 7,041,293; 7,038,018; 7,037,498; 7,012,133; 7,001,598;6,998,468; 6,994,976; 6,994,852; 6,989,241; 6,974,863; 6,965,018;6,964,854; 6,962,981; 6,962,813; 6,956,107; 6,951,924; 6,949,244;6,946,129; 6,943,020; 6,939,547; 6,921,645; 6,921,645; 6,921,533;6,919,433; 6,919,078; 6,916,475; 6,905,681; 6,899,879; 6,893,625;6,887,468; 6,887,466; 6,884,594; 6,881,405; 6,878,812; 6,875,580;6,872,568; 6,867,006; 6,864,062; 6,861,511; 6,861,227; 6,861,226;6,838,282; 6,835,549; 6,835,370; 6,824,780; 6,824,778; 6,812,206;6,793,924; 6,783,758; 6,770,450; 6,767,711; 6,764,688; 6,764,681;6,764,679; 6,743,898; 6,733,981; 6,730,307; 6,720,155; 6,716,966;6,709,653; 6,693,176; 6,692,908; 6,689,607; 6,689,362; 6,689,355;6,682,737; 6,682,736; 6,682,734; 6,673,344; 6,653,104; 6,652,852;6,635,482; 6,630,144; 6,610,833; 6,610,294; 6,605,441; 6,605,279;6,596,852; 6,592,868; 6,576,745; 6,572,856; 6,566,076; 6,562,618;6,545,130; 6,544,749; 6,534,058; 6,528,625; 6,528,269; 6,521,227;6,518,404; 6,511,665; 6,491,915; 6,488,930; 6,482,598; 6,482,408;6,479,247; 6,468,531; 6,468,529; 6,465,173; 6,461,823; 6,458,356;6,455,044; 6,455,040, 6,451,310; 6,444,206; 6,441,143; 6,432,404;6,432,402; 6,419,928; 6,413,726; 6,406,694; 6,403,770; 6,403,091;6,395,276; 6,395,274; 6,387,350; 6,383,759; 6,383,484; 6,376,654;6,372,215; 6,359,126; 6,355,481; 6,355,444; 6,355,245; 6,355,244;6,346,246; 6,344,198; 6,340,571; 6,340,459; 6,331,175; 6,306,393;6,254,868; 6,187,287; 6,183,744; 6,129,914; 6,120,767; 6,096,289;6,077,499; 5,922,302; 5,874,540; 5,814,440; 5,798,229; 5,789,554;5,776,456; 5,736,119; 5,716,595; 5,677,136; 5,587,459; 5,443,953,5,525,338, the Examples section of each of which is incorporated hereinby reference. These are exemplary only and a wide variety of otherantibodies and their hybridomas are known in the art. The skilledartisan will realize that antibody sequences or antibody-secretinghybridomas against almost any disease-associated antigen may be obtainedby a simple search of the ATCC, NCBI and/or USPTO databases forantibodies against a selected disease-associated target of interest. Theantigen binding domains of the cloned antibodies may be amplified,excised, ligated into an expression vector, transfected into an adaptedhost cell and used for protein production, using standard techniqueswell known in the art. Isolated antibodies may be conjugated totherapeutic agents that induce DNA strand breaks, such as camptothecinsor anthracyclines, using the techniques disclosed herein.

Chimeric and Humanized Antibodies

A chimeric antibody is a recombinant protein in which the variableregions of a human antibody have been replaced by the variable regionsof, for example, a mouse antibody, including thecomplementarity-determining regions (CDRs) of the mouse antibody.Chimeric antibodies exhibit decreased immunogenicity and increasedstability when administered to a subject. Methods for constructingchimeric antibodies are well known in the art (e.g., Leung et al., 1994,Hybridoma 13:469).

A chimeric monoclonal antibody may be humanized by transferring themouse CDRs from the heavy and light variable chains of the mouseimmunoglobulin into the corresponding variable domains of a humanantibody. The mouse framework regions (FR) in the chimeric monoclonalantibody are also replaced with human FR sequences. To preserve thestability and antigen specificity of the humanized monoclonal, one ormore human FR residues may be replaced by the mouse counterpartresidues. Humanized monoclonal antibodies may be used for therapeutictreatment of subjects. Techniques for production of humanized monoclonalantibodies are well known in the art. (See, e.g., Jones et al., 1986,Nature, 321:522; Riechmann et al., Nature, 1988, 332:323; Verhoeyen etal., 1988, Science, 239:1534; Carter et al., 1992, Proc. Nat'l Acad.Sci. USA, 89:4285; Sandhu, Crit. Rev. Biotech., 1992, 12:437; Tempest etal., 1991, Biotechnology 9:266; Singer et al., J. Immun., 1993,150:2844.)

Other embodiments may concern non-human primate antibodies. Generaltechniques for raising therapeutically useful antibodies in baboons maybe found, for example, in Goldenberg et al., WO 91/11465 (1991), and inLosman et al., Int. J. Cancer 46: 310 (1990). In another embodiment, anantibody may be a human monoclonal antibody. Such antibodies may beobtained from transgenic mice that have been engineered to producespecific human antibodies in response to antigenic challenge, asdiscussed below.

Human Antibodies

Methods for producing fully human antibodies using either combinatorialapproaches or transgenic animals transformed with human immunoglobulinloci are known in the art (e.g., Mancini et al., 2004, New Microbiol.27:315-28; Conrad and Scheller, 2005, Comb. Chem. High ThroughputScreen. 8:117-26; Brekke and Loset, 2003, Curr. Opin. Phamacol.3:544-50; each incorporated herein by reference). Such fully humanantibodies are expected to exhibit even fewer side effects than chimericor humanized antibodies and to function in vivo as essentiallyendogenous human antibodies. In certain embodiments, the claimed methodsand procedures may utilize human antibodies produced by such techniques.

In one alternative, the phage display technique may be used to generatehuman antibodies (e.g., Dantas-Barbosa et al., 2005, Genet. Mol. Res.4:126-40, incorporated herein by reference). Human antibodies may begenerated from normal humans or from humans that exhibit a particulardisease state, such as cancer (Dantas-Barbosa et al., 2005). Theadvantage to constructing human antibodies from a diseased individual isthat the circulating antibody repertoire may be biased towardsantibodies against disease-associated antigens.

In one non-limiting example of this methodology, Dantas-Barbosa et al.(2005) constructed a phage display library of human Fab antibodyfragments from osteosarcoma patients. Generally, total RNA was obtainedfrom circulating blood lymphocytes (Id.) Recombinant Fab were clonedfrom the μ, γ and κ chain antibody repertoires and inserted into a phagedisplay library (Id.) RNAs were converted to cDNAs and used to make FabcDNA libraries using specific primers against the heavy and light chainimmunoglobulin sequences (Marks et al., 1991, J. Mol. Biol. 222:581-97,incorporated herein by reference). Library construction was performedaccording to Andris-Widhopf et al. (2000, In: Phage Display LaboratoryManual, Barbas et al. (eds), 1^(st) edition, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. pp. 9.1 to 9.22, incorporatedherein by reference). The final Fab fragments were digested withrestriction endonucleases and inserted into the bacteriophage genome tomake the phage display library. Such libraries may be screened bystandard phage display methods. The skilled artisan will realize thatthis technique is exemplary only and any known method for making andscreening human antibodies or antibody fragments by phage display may beutilized.

In another alternative, transgenic animals that have been geneticallyengineered to produce human antibodies may be used to generateantibodies against essentially any immunogenic target, using standardimmunization protocols as discussed above. Methods for obtaining humanantibodies from transgenic mice are described by Green et al., NatureGenet. 7:13 (1994), Lonberg et al., Nature 368:856 (1994), and Taylor etal., Int. Immun. 6:579 (1994). A non-limiting example of such a systemis the XENOMOUSE® (e.g., Green et al., 1999, J. Immunol. Methods231:11-23, incorporated herein by reference) from Abgenix (Fremont,Calif.). In the XENOMOUSE® and similar animals, the mouse antibody geneshave been inactivated and replaced by functional human antibody genes,while the remainder of the mouse immune system remains intact.

The XENOMOUSE® was transformed with germline-configured YACs (yeastartificial chromosomes) that contained portions of the human IgH and Igkappa loci, including the majority of the variable region sequences,along accessory genes and regulatory sequences. The human variableregion repertoire may be used to generate antibody producing B cells,which may be processed into hybridomas by known techniques. A XENOMOUSE®immunized with a target antigen will produce human antibodies by thenormal immune response, which may be harvested and/or produced bystandard techniques discussed above. A variety of strains of XENOMOUSE®are available, each of which is capable of producing a different classof antibody. Transgenically produced human antibodies have been shown tohave therapeutic potential, while retaining the pharmacokineticproperties of normal human antibodies (Green et al., 1999). The skilledartisan will realize that the claimed compositions and methods are notlimited to use of the XENOMOUSE® system but may utilize any transgenicanimal that has been genetically engineered to produce human antibodies.

Production of Antibody Fragments

Some embodiments of the claimed methods and/or compositions may concernantibody fragments. Such antibody fragments may be obtained, forexample, by pepsin or papain digestion of whole antibodies byconventional methods. For example, antibody fragments may be produced byenzymatic cleavage of antibodies with pepsin to provide a 5S fragmentdenoted F(ab′)₂. This fragment may be further cleaved using a thiolreducing agent and, optionally, a blocking group for the sulfhydrylgroups resulting from cleavage of disulfide linkages, to produce 3.5SFab′ monovalent fragments. Alternatively, an enzymatic cleavage usingpepsin produces two monovalent Fab fragments and an Fc fragment.Exemplary methods for producing antibody fragments are disclosed in U.S.Pat. Nos. 4,036,945; 4,331,647; Nisonoff et al., 1960, Arch. Biochem.Biophys., 89:230; Porter, 1959, Biochem. J., 73:119; Edelman et al.,1967, METHODS IN ENZYMOLOGY, page 422 (Academic Press), and Coligan etal. (eds.), 1991, CURRENT PROTOCOLS IN IMMUNOLOGY, (John Wiley & Sons).

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments or other enzymatic, chemical or genetic techniques also may beused, so long as the fragments bind to the antigen that is recognized bythe intact antibody. For example, Fv fragments comprise an associationof V_(H) and V_(L) chains. This association can be noncovalent, asdescribed in Inbar et al., 1972, Proc. Nat'l. Acad. Sci. USA, 69:2659.Alternatively, the variable chains may be linked by an intermoleculardisulfide bond or cross-linked by chemicals such as glutaraldehyde. SeeSandhu, 1992, Crit. Rev. Biotech., 12:437.

Preferably, the Fv fragments comprise V_(H) and V_(L) chains connectedby a peptide linker. These single-chain antigen binding proteins (scFv)are prepared by constructing a structural gene comprising DNA sequencesencoding the V_(H) and V_(L) domains, connected by an oligonucleotideslinker sequence. The structural gene is inserted into an expressionvector that is subsequently introduced into a host cell, such as E.coli. The recombinant host cells synthesize a single polypeptide chainwith a linker peptide bridging the two V domains. Methods for producingscFvs are well-known in the art. See Whitlow et al., 1991, Methods: ACompanion to Methods in Enzymology 2:97; Bird et al., 1988, Science,242:423; U.S. Pat. No. 4,946,778; Pack et al., 1993, Bio/Technology,11:1271, and Sandhu, 1992, Crit. Rev. Biotech., 12:437.

Another form of an antibody fragment is a single-domain antibody (dAb),sometimes referred to as a single chain antibody. Techniques forproducing single-domain antibodies are well known in the art (see, e.g.,Cossins et al., Protein Expression and Purification, 2007, 51:253-59;Shuntao et al., Molec Immunol 2006, 43:1912-19; Tanha et al., J. Biol.Chem. 2001, 276:24774-780). Other types of antibody fragments maycomprise one or more complementarity-determining regions (CDRs). CDRpeptides (“minimal recognition units”) can be obtained by constructinggenes encoding the CDR of an antibody of interest. Such genes areprepared, for example, by using the polymerase chain reaction tosynthesize the variable region from RNA of antibody-producing cells. SeeLarrick et al., 1991, Methods: A Companion to Methods in Enzymology2:106; Ritter et al. (eds.), 1995, MONOCLONAL ANTIBODIES: PRODUCTION,ENGINEERING AND CLINICAL APPLICATION, pages 166-179 (CambridgeUniversity Press); Birch et al., (eds.), 1995, MONOCLONAL ANTIBODIES:PRINCIPLES AND APPLICATIONS, pages 137-185 (Wiley-Liss, Inc.)

Antibody Variations

In certain embodiments, the sequences of antibodies, such as the Fcportions of antibodies, may be varied to optimize the physiologicalcharacteristics of the conjugates, such as the half-life in serum.Methods of substituting amino acid sequences in proteins are widelyknown in the art, such as by site-directed mutagenesis (e.g. Sambrook etal., Molecular Cloning, A laboratory manual, 2^(nd) Ed, 1989). Inpreferred embodiments, the variation may involve the addition or removalof one or more glycosylation sites in the Fc sequence (e.g., U.S. Pat.No. 6,254,868, the Examples section of which is incorporated herein byreference). In other preferred embodiments, specific amino acidsubstitutions in the Fc sequence may be made (e.g., Hornick et al.,2000, J Nucl Med 41:355-62; Hinton et al., 2006, J Immunol 176:346-56;Petkova et al. 2006, Int Immunol 18:1759-69; U.S. Pat. No. 7,217,797;each incorporated herein by reference).

Target Antigens and Exemplary Antibodies

In a preferred embodiment, antibodies are used that recognize and/orbind to human antigens that are expressed at high levels on target cellsand that are expressed predominantly or exclusively on diseased cellsversus normal tissues. More preferably, the antibodies internalizerapidly following binding. An exemplary rapidly internalizing antibodyis the LL1 (anti-CD74) antibody, with a rate of internalization ofapproximately 8×10⁶ antibody molecules per cell per day (e.g., Hansen etal., 1996, Biochem J. 320:293-300). Thus, a “rapidly internalizing”antibody may be one with an internalization rate of about 1×10⁶ to about1×10⁷ antibody molecules per cell per day. Antibodies of use in theclaimed compositions and methods may include MAbs with properties asrecited above. Exemplary antibodies of use for therapy of, for example,cancer include but are not limited to LL1 (anti-CD74), LL2 or RFB4(anti-CD22), veltuzumab (hA20, anti-CD20), rituxumab (anti-CD20),obinutuzumab (GA101, anti-CD20), lambrolizumab (anti-PD-1 receptor),nivolumab (anti-PD-1 receptor), ipilimumab (anti-CTLA-4), RS7(anti-epithelial glycoprotein-1 (EGP-1, also known as Trop-2)), PAM4 orKC4 (both anti-mucin), MN-14 (anti-carcinoembryonic antigen (CEA, alsoknown as CD66e or CEACAM5), MN-15 or MN-3 (anti-CEACAM6), Mu-9(anti-colon-specific antigen-p), Immu 31 (an anti-alpha-fetoprotein), R1(anti-IGF-1R), A19 (anti-CD19), TAG-72 (e.g., CC49), Tn, J591 or HuJ591(anti-PSMA (prostate-specific membrane antigen)), AB-PG1-XG1-026(anti-PSMA dimer), D2/B (anti-PSMA), G250 (an anti-carbonic anhydrase IXMAb), L243 (anti-HLA-DR) alemtuzumab (anti-CD52), bevacizumab(anti-VEGF), cetuximab (anti-EGFR), gemtuzumab (anti-CD33), ibritumomabtiuxetan (anti-CD20); panitumumab (anti-EGFR); tositumomab (anti-CD20);PAM4 (aka clivatuzumab, anti-mucin) and trastuzumab (anti-ErbB2). Suchantibodies are known in the art (e.g., U.S. Pat. Nos. 5,686,072;5,874,540; 6,107,090; 6,183,744; 6,306,393; 6,653,104; 6,730.300;6,899,864; 6,926,893; 6,962,702; 7,074,403; 7,230,084; 7,238,785;7,238,786; 7,256,004; 7,282,567; 7,300,655; 7,312,318; 7,585,491;7,612,180; 7,642,239; and U.S. Patent Application Publ. No. 20050271671;20060193865; 20060210475; 20070087001; the Examples section of eachincorporated herein by reference.) Specific known antibodies of useinclude hPAM4 (U.S. Pat. No. 7,282,567), hA20 (U.S. Pat. No. 7,151,164),hA19 (U.S. Pat. No. 7,109,304), hIMMU-31 (U.S. Pat. No. 7,300,655), hLL1(U.S. Pat. No. 7,312,318), hLL2 (U.S. Pat. No. 5,789,554), hMu-9 (U.S.Pat. No. 7,387,772), hL243 (U.S. Pat. No. 7,612,180), hMN-14 (U.S. Pat.No. 6,676,924), hMN-15 (U.S. Pat. No. 8,287,865), hR1 (U.S. patentapplication Ser. No. 14/061,1767), hRS7 (U.S. Pat. No. 7,238,785), hMN-3(U.S. Pat. No. 7,541,440), AB-PG1-XG1-026 (U.S. patent application Ser.No. 11/983,372, deposited as ATCC PTA-4405 and PTA-4406) and D2/B (WO2009/130575) the text of each recited patent or application isincorporated herein by reference with respect to the Figures andExamples sections.

Other useful antigens that may be targeted using the describedconjugates include carbonic anhydrase IX, B7, CCL19, CCL21, CSAp,HER-2/neu, BrE3, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15,CD16, CD18, CD19, CD20 (e.g., C2B8, hA20, 1F5 MAbs), CD21, CD22, CD23,CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD44, CD45,CD46, CD52, CD54, CD55, CD59, CD64, CD67, CD70, CD74, CD79a, CD80, CD83,CD95, CD126, CD133, CD138, CD147, CD154, CEACAM5, CEACAM6, CTLA-4,alpha-fetoprotein (AFP), VEGF (e.g., AVASTIN®, fibronectin splicevariant), ED-B fibronectin (e.g., L19), EGP-1 (Trop-2), EGP-2 (e.g.,17-1A), EGF receptor (ErbB1) (e.g., ERBITUX®), ErbB2, ErbB3, Factor H,FHL-1, Flt-3, folate receptor, Ga 733,GRO-β, HMGB-1, hypoxia induciblefactor (HIF), HM1.24, HER-2/neu, insulin-like growth factor (ILGF),IFN-γ, IFN-α, IFN-β, IFN-λ, IL-2R, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R,IL-18R, IL-2, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-25, IP-10,IGF-1R, Ia, HM1.24, gangliosides, HCG, the HLA-DR antigen to which L243binds, CD66 antigens, i.e., CD66a-d or a combination thereof, MAGE,mCRP, MCP-1, MIP-1A, MIP-1B, macrophage migration-inhibitory factor(MIF), MUC1, MUC2, MUC3, MUC4, MUC5ac, placental growth factor (P1GF),PSA (prostate-specific antigen), PSMA, PAM4 antigen, PD-1 receptor,NCA-95, NCA-90, A3, A33, Ep-CAM, KS-1, Le(y), mesothelin, S100,tenascin, TAC, Tn antigen, Thomas-Friedenreich antigens, tumor necrosisantigens, tumor angiogenesis antigens, TNF-α, TRAIL receptor (R1 andR2), Trop-2, VEGFR, RANTES, T101, as well as cancer stem cell antigens,complement factors C3, C3a, C3b, C5a, C5, and an oncogene product.

A comprehensive analysis of suitable antigen (Cluster Designation, orCD) targets on hematopoietic malignant cells, as shown by flow cytometryand which can be a guide to selecting suitable antibodies fordrug-conjugated immunotherapy, is Craig and Foon, Blood prepublishedonline Jan. 15, 2008; DOL 10.1182/blood-2007-11-120535.

The CD66 antigens consist of five different glycoproteins with similarstructures, CD66a-e, encoded by the carcinoembryonic antigen (CEA) genefamily members, BCG, CGM6, NCA, CGM1 and CEA, respectively. These CD66antigens (e.g., CEACAM6) are expressed mainly in granulocytes, normalepithelial cells of the digestive tract and tumor cells of varioustissues. Also included as suitable targets for cancers are cancer testisantigens, such as NY-ESO-1 (Theurillat et al., Int. J Cancer 2007;120(11):2411-7), as well as CD79a in myeloid leukemia (Kozlov et al.,Cancer Genet. Cytogenet. 2005; 163(1):62-7) and also B-cell diseases,and CD79b for non-Hodgkin's lymphoma (Poison et al., Blood110(2):616-623). A number of the aforementioned antigens are disclosedin U.S. Provisional Application Ser. No. 60/426,379, entitled “Use ofMulti-specific, Non-covalent Complexes for Targeted Delivery ofTherapeutics,” filed Nov. 15, 2002. Cancer stem cells, which areascribed to be more therapy-resistant precursor malignant cellpopulations (Hill and Perris, J. Natl. Cancer Inst. 2007; 99:1435-40),have antigens that can be targeted in certain cancer types, such asCD133 in prostate cancer (Maitland et al., Ernst Schering Found. Sympos.Proc. 2006; 5:155-79), non-small-cell lung cancer (Donnenberg et al., J.Control Release 2007; 122(3):385-91), and glioblastoma (Beier et al.,Cancer Res. 2007; 67(9):4010-5), and CD44 in colorectal cancer (Dalerbaet al., Proc. Natl. Acad. Sci. USA 2007; 104(24)10158-63), pancreaticcancer (Li et al., Cancer Res. 2007; 67(3):1030-7), and in head and necksquamous cell carcinoma (Prince et al., Proc. Natl. Acad. Sci. USA 2007;104(3)973-8).

For multiple myeloma therapy, suitable targeting antibodies have beendescribed against, for example, CD38 and CD138 (Stevenson, Mol Med 2006;12(11-12):345-346; Tassone et al., Blood 2004; 104(12):3688-96), CD74(Stein et al., ibid.), CS1 (Tai et al., Blood 2008; 112(4):1329-37, andCD40 (Tai et al., 2005; Cancer Res. 65(13):5898-5906).

Macrophage migration inhibitory factor (MIF) is an important regulatorof innate and adaptive immunity and apoptosis. It has been reported thatCD74 is the endogenous receptor for MIF (Leng et al., 2003, J Exp Med197:1467-76). The therapeutic effect of antagonistic anti-CD74antibodies on MIF-mediated intracellular pathways may be of use fortreatment of a broad range of disease states, such as cancers of thebladder, prostate, breast, lung, colon and chronic lymphocytic leukemia(e.g., Meyer-Siegler et al., 2004, BMC Cancer 12:34; Shachar & Haran,2011, Leuk Lymphoma 52:1446-54); autoimmune diseases such as rheumatoidarthritis and systemic lupus erythematosus (Morand & Leech, 2005, FrontBiosci 10:12-22; Shachar & Haran, 2011, Leuk Lymphoma 52:1446-54);kidney diseases such as renal allograft rejection (Lan, 2008, NephronExp Nephrol. 109:e79-83); and numerous inflammatory diseases(Meyer-Siegler et al., 2009, Mediators Inflamm epub Mar. 22, 2009;Takahashi et al., 2009, Respir Res 10:33; Milatuzumab (hLL1) is anexemplary anti-CD74 antibody of therapeutic use for treatment ofMIF-mediated diseases.

Anti-TNF-α antibodies are known in the art and may be of use to treatimmune diseases, such as autoimmune disease, immune dysfunction (e.g.,graft-versus-host disease, organ transplant rejection) or diabetes.Known antibodies against TNF-α include the human antibody CDP571 (Ofeiet al., 2011, Diabetes 45:881-85); murine antibodies MTNFAI, M2TNFAI,M3TNFAI, M3TNFABI, M302B and M303 (Thermo Scientific, Rockford, Ill.);infliximab (Centocor, Malvern, Pa.); certolizumab pegol (UCB, Brussels,Belgium); and adalimumab (Abbott, Abbott Park, Ill.). These and manyother known anti-TNF-α antibodies may be used in the claimed methods andcompositions. Other antibodies of use for therapy of immunedysregulatory or autoimmune disease include, but are not limited to,anti-B-cell antibodies such as veltuzumab, epratuzumab, milatuzumab orhL243; tocilizumab (anti-IL-6 receptor); basiliximab (anti-CD25);daclizumab (anti-CD25); efalizumab (anti-CD11a); muromonab-CD3 (anti-CD3receptor); anti-CD40L (UCB, Brussels, Belgium); natalizumab (anti-α4integrin) and omalizumab (anti-IgE).

Type-1 and Type-2 diabetes may be treated using known antibodies againstB-cell antigens, such as CD22 (epratuzumab and hRFB4), CD74(milatuzumab), CD19 (hA19), CD20 (veltuzumab) or HLA-DR (hL243) (see,e.g., Winer et al., 2011, Nature Med 17:610-18). Anti-CD3 antibodiesalso have been proposed for therapy of type 1 diabetes (Cernea et al.,2010, Diabetes Metab Rev 26:602-05).

In another preferred embodiment, antibodies are used that internalizerapidly and are then re-expressed, processed and presented on cellsurfaces, enabling continual uptake and accretion of circulatingconjugate by the cell. An example of a most-preferred antibody/antigenpair is LL1, an anti-CD74 MAb (invariant chain, class II-specificchaperone, Ii) (see, e.g., U.S. Pat. Nos. 6,653,104; 7,312,318; theExamples section of each incorporated herein by reference). The CD74antigen is highly expressed on B-cell lymphomas (including multiplemyeloma) and leukemias, certain T-cell lymphomas, melanomas, colonic,lung, and renal cancers, glioblastomas, and certain other cancers (Onget al., Immunology 98:296-302 (1999)). A review of the use of CD74antibodies in cancer is contained in Stein et al., Clin Cancer Res. 2007Sep. 15; 13(18 Pt 2):55565-5563s, incorporated herein by reference.

The diseases that are preferably treated with anti-CD74 antibodiesinclude, but are not limited to, non-Hodgkin's lymphoma, Hodgkin'sdisease, melanoma, lung, renal, colonic cancers, glioblastomemultiforme, histiocytomas, myeloid leukemias, and multiple myeloma.Continual expression of the CD74 antigen for short periods of time onthe surface of target cells, followed by internalization of the antigen,and re-expression of the antigen, enables the targeting LL1 antibody tobe internalized along with any drug moiety it carries. This allows ahigh, and therapeutic, concentration of LL1-drug conjugate to beaccumulated inside such cells. Internalized LL1-drug conjugates arecycled through lysosomes and endosomes, and the drug moiety is releasedin an active form within the target cells.

Antibodies of use to treat autoimmune disease or immune systemdysfunctions (e.g., graft-versus-host disease, organ transplantrejection) are known in the art and may be conjugated to SN-38 using thedisclosed methods and compositions. Antibodies of use to treatautoimmune/immune dysfunction disease may bind to exemplary antigensincluding, but not limited to, BCL-1, BCL-2, BCL-6, CD1a, CD2, CD3, CD4,CD5, CD7, CD8, CD10, CD11b, CD11c, CD13, CD14, CD15, CD16, CD19, CD20,CD21, CD22, CD23, CD25, CD33, CD34, CD38, CD40, CD40L, CD41a, CD43,CD45, CD55, CD74, TNF-alpha, interferon and HLA-DR. Antibodies that bindto these and other target antigens, discussed above, may be used totreat autoimmune or immune dysfunction diseases. Autoimmune diseasesthat may be treated with antibodies or immunoconjugates may includeacute idiopathic thrombocytopenic purpura, chronic idiopathicthrombocytopenic purpura, dermatomyositis, Sydenham's chorea, myastheniagravis, systemic lupus erythematosus, lupus nephritis, rheumatic fever,polyglandular syndromes, bullous pemphigoid, diabetes mellitus,Henoch-Schonlein purpura, post-streptococcal nephritis, erythemanodosum, Takayasu's arteritis, ANCA-associated vasculitides, Addison'sdisease, rheumatoid arthritis, multiple sclerosis, sarcoidosis,ulcerative colitis, erythema multiforme, IgA nephropathy, polyarteritisnodosa, ankylosing spondylitis, Goodpasture's syndrome, thromboangitisobliterans, Sjogren's syndrome, primary biliary cirrhosis, Hashimoto'sthyroiditis, thyrotoxicosis, scleroderma, chronic active hepatitis,polymyositis/dermatomyositis, polychondritis, bullous pemphigoid,pemphigus vulgaris, Wegener's granulomatosis, membranous nephropathy,amyotrophic lateral sclerosis, tabes dorsalis, giant cellarteritis/polymyalgia, pernicious anemia, rapidly progressiveglomerulonephritis, psoriasis or fibrosing alveolitis.

The antibodies discussed above and other known antibodies againstdisease-associated antigens may be used as CPT-conjugates, morepreferably SN-38-conjugates, in the practice of the claimed methods andcompositions.

Bispecific and Multispecific Antibodies

Bispecific antibodies are useful in a number of biomedical applications.For instance, a bispecific antibody with binding sites for a tumor cellsurface antigen and for a T-cell surface receptor can direct the lysisof specific tumor cells by T cells. Bispecific antibodies recognizinggliomas and the CD3 epitope on T cells have been successfully used intreating brain tumors in human patients (Nitta, et al. Lancet. 1990;355:368-371). A preferred bispecific antibody is an anti-CD3 X anti-CD19antibody. In alternative embodiments, an anti-CD3 antibody or fragmentthereof may be attached to an antibody or fragment against anotherB-cell associated antigen, such as anti-CD3 X anti-CD20, anti-CD3 Xanti-CD22, anti-CD3 X anti-HLA-DR or anti-CD3 X anti-CD74. In certainembodiments, the techniques and compositions for therapeutic agentconjugation disclosed herein may be used with bispecific ormultispecific antibodies as the targeting moieties.

Numerous methods to produce bispecific or multispecific antibodies areknown, as disclosed, for example, in U.S. Pat. No. 7,405,320, theExamples section of which is incorporated herein by reference.Bispecific antibodies can be produced by the quadroma method, whichinvolves the fusion of two different hybridomas, each producing amonoclonal antibody recognizing a different antigenic site (Milstein andCuello, Nature, 1983; 305:537-540).

Another method for producing bispecific antibodies usesheterobifunctional cross-linkers to chemically tether two differentmonoclonal antibodies (Staerz, et al. Nature, 1985; 314:628-631; Perez,et al. Nature, 1985; 316:354-356). Bispecific antibodies can also beproduced by reduction of each of two parental monoclonal antibodies tothe respective half molecules, which are then mixed and allowed toreoxidize to obtain the hybrid structure (Staerz and Bevan. Proc NatlAcad Sci USA. 1986; 83:1453-1457). Another alternative involveschemically cross-linking two or three separately purified Fab′ fragmentsusing appropriate linkers. (See, e.g., European Patent Application0453082).

Other methods include improving the efficiency of generating hybridhybridomas by gene transfer of distinct selectable markers viaretrovirus-derived shuttle vectors into respective parental hybridomas,which are fused subsequently (DeMonte, et al. Proc Natl Acad Sci USA.1990, 87:2941-2945); or transfection of a hybridoma cell line withexpression plasmids containing the heavy and light chain genes of adifferent antibody.

Cognate V_(H) and V_(L) domains can be joined with a peptide linker ofappropriate composition and length (usually consisting of more than 12amino acid residues) to form a single-chain Fv (scFv) with bindingactivity. Methods of manufacturing scFvs are disclosed in U.S. Pat. Nos.4,946,778 and 5,132,405, the Examples section of each of which isincorporated herein by reference. Reduction of the peptide linker lengthto less than 12 amino acid residues prevents pairing of V_(H) and V_(L)domains on the same chain and forces pairing of V_(H) and V_(L) domainswith complementary domains on other chains, resulting in the formationof functional multimers. Polypeptide chains of V_(H) and V_(L) domainsthat are joined with linkers between 3 and 12 amino acid residues formpredominantly dimers (termed diabodies). With linkers between 0 and 2amino acid residues, trimers (termed triabody) and tetramers (termedtetrabody) are favored, but the exact patterns of oligomerization appearto depend on the composition as well as the orientation of V-domains(V_(H)-linker-V_(L) or V_(L)-linker-V_(H)), in addition to the linkerlength.

These techniques for producing multispecific or bispecific antibodiesexhibit various difficulties in terms of low yield, necessity forpurification, low stability or the labor-intensiveness of the technique.More recently, a technique known as “dock and lock” (DNL) has beenutilized to produce combinations of virtually any desired antibodies,antibody fragments and other effector molecules (see, e.g., U.S. Pat.Nos. 7,521,056; 7,527,787; 7,534,866; 7,550,143; 7,666,400; 7,858,070;7,871,622; 7,906,121; 7,906,118; 8,163,291; 7,901,680; 7,981,398;8,003,111 and 8,034,352, the Examples section of each of whichincorporated herein by reference). The technique utilizes complementaryprotein binding domains, referred to as anchoring domains (AD) anddimerization and docking domains (DDD), which bind to each other andallow the assembly of complex structures, ranging from dimers, trimers,tetramers, quintamers and hexamers. These form stable complexes in highyield without requirement for extensive purification. The DNL techniqueallows the assembly of monospecific, bispecific or multispecificantibodies. Any of the techniques known in the art for making bispecificor multispecific antibodies may be utilized in the practice of thepresently claimed methods.

In various embodiments, a conjugate as disclosed herein may be part of acomposite, multispecific antibody. Such antibodies may contain two ormore different antigen binding sites, with differing specificities. Themultispecific composite may bind to different epitopes of the sameantigen, or alternatively may bind to two different antigens. Some ofthe more preferred target combinations include those listed in Table 1.This is a list of examples of preferred combinations, but is notintended to be exhaustive.

TABLE 1 Some Examples of multispecific antibodies. First target Secondtarget MIF A second proinflammatory effector cytokine, especiallyHMGB-1, TNF-α, IL-1, or IL-6 MIF Proinflammatory effector chemokine,especially MCP-1, RANTES, MIP-1A, or MIP-1B MIF Proinflammatory effectorreceptor, especially IL-6R, IL-13R, and IL-15R MIF Coagulation factor,especially TF or thrombin MIF Complement factor, especially C3, C5, C3a,or C5a MIF Complement regulatory protein, especially CD46, CD55, CD59,and mCRP MIF Cancer associated antigen or receptor HMGB-1 A secondproinflammatory effector cytokine, especially MIF, TNF-α, IL-1, or IL-6HMGB-1 Proinflammatory effector chemokine, especially MCP-1, RANTES,MIP-1A, or MIP-1B HMGB-1 Proinflammatory effector receptor especiallyMCP-1, RANTES, MIP-1A, or MIP-1B HMGB-1 Coagulation factor, especiallyTF or thrombin HMGB-1 Complement factor, especially C3, C5, C3a, or C5aHMGB-1 Complement regulatory protein, especially CD46, CD55, CD59, andmCRP HMGB-1 Cancer associated antigen or receptor TNF-α A secondproinflammatory effector cytokine, especially MIF, HMGB-1, TNF-α, IL-1,or IL-6 TNF-α Proinflammatory effector chemokine, especially MCP-1,RANTES, MIP-1A, or MIP-1B TNF-α Proinflammatory effector receptor,especially IL-6R IL-13R, and IL-15R TNF-α Coagulation factor, especiallyTF or thrombin TNF-α Complement factor, especially C3, C5, C3a, or C5aTNF-α Complement regulatory protein, especially CD46, CD55, CD59, andmCRP TNF-α Cancer associated antigen or receptor LPS Proinflammatoryeffector cytokine, especially MIF, HMGB-1, TNF-α, IL-1, or IL-6 LPSProinflammatory effector chemokine, especially MCP-1, RANTES, MIP-1A, orMIP-1B LPS Proinflammatory effector receptor, especially IL-6R IL-13R,and IL-15R LPS Coagulation factor, especially TF or thrombin LPSComplement factor, especially C3, C5, C3a, or C5a LPS Complementregulatory protein, especially CD46, CD55, CD59, and mCRP TF or thrombinProinflammatory effector cytokine, especially MIF, HMGB-1, TNF-α, IL-1,or IL-6 TF or thrombin Proinflammatory effector chemokine, especiallyMCP-1, RANTES, MIP-1A, or MIP-1B TF or thrombin Proinflammatory effectorreceptor, especially IL-6R IL-13R, and IL-15R TF or thrombin Complementfactor, especially C3, C5, C3a, or C5a TF or thrombin Complementregulatory protein, especially CD46, CD55, CD59, and mCRP TF or thrombinCancer associated antigen or receptor

Still other combinations, such as are preferred for cancer therapies,include CD20+CD22 antibodies, CD74+CD20 antibodies, CD74+CD22antibodies, CEACAM5 (CEA)+CEACAM6 (NCA) antibodies, insulin-like growthfactor (ILGF)+CEACAM5 antibodies, EGP-1 (e.g., RS-7)+ILGF antibodies,CEACAM5+EGFR antibodies, IL6+CEACAM6 antibodies, CD74 and HLA-DRantibodies, and CD22 and HLA-DR antibodies. Such antibodies need notonly be used in combination, but can be combined as fusion proteins ofvarious forms, such as IgG, Fab, scFv, and the like, as described inU.S. Pat. Nos. 6,083,477; 6,183,744 and 6,962,702 and U.S. PatentApplication Publication Nos. 20030124058; 20030219433; 20040001825;20040202666; 20040219156; 20040219203; 20040235065; 20050002945;20050014207; 20050025709; 20050079184; 20050169926; 20050175582;20050249738; 20060014245 and 20060034759, the Examples section of eachincorporated herein by reference.

DOCK-AND-LOCK® (DNL®)

In preferred embodiments, a bivalent or multivalent antibody is formedas a DOCK-AND-LOCK® (DNL®) complex (see, e.g., U.S. Pat. Nos. 7,521,056;7,527,787; 7,534,866; 7,550,143; 7,666,400; 7,858,070; 7,871,622;7,906,121; 7,906,118; 8,163,291; 7,901,680; 7,981,398; 8,003,111 and8,034,352, the Examples section of each of which is incorporated hereinby reference.) Generally, the technique takes advantage of the specificand high-affinity binding interactions that occur between a dimerizationand docking domain (DDD) sequence of the regulatory (R) subunits ofcAMP-dependent protein kinase (PKA) and an anchor domain (AD) sequencederived from any of a variety of AKAP proteins (Baillie et al., FEBSLetters. 2005; 579: 3264. Wong and Scott, Nat. Rev. Mol. Cell Biol.2004; 5: 959). The DDD and AD peptides may be attached to any protein,peptide or other molecule. Because the DDD sequences spontaneouslydimerize and bind to the AD sequence, the technique allows the formationof complexes between any selected molecules that may be attached to DDDor AD sequences.

Although the standard DNL® complex comprises a trimer with twoDDD-linked molecules attached to one AD-linked molecule, variations incomplex structure allow the formation of dimers, trimers, tetramers,pentamers, hexamers and other multimers. In some embodiments, the DNL®complex may comprise two or more antibodies, antibody fragments orfusion proteins which bind to the same antigenic determinant or to twoor more different antigens. The DNL® complex may also comprise one ormore other effectors, such as proteins, peptides, immunomodulators,cytokines, interleukins, interferons, binding proteins, peptide ligands,carrier proteins, toxins, ribonucleases such as onconase, inhibitoryoligonucleotides such as siRNA, antigens or xenoantigens, polymers suchas PEG, enzymes, therapeutic agents, hormones, cytotoxic agents,anti-angiogenic agents, pro-apoptotic agents or any other molecule oraggregate.

PKA, which plays a central role in one of the best studied signaltransduction pathways triggered by the binding of the second messengercAMP to the R subunits, was first isolated from rabbit skeletal musclein 1968 (Walsh et al., J. Biol. Chem. 1968; 243:3763). The structure ofthe holoenzyme consists of two catalytic subunits held in an inactiveform by the R subunits (Taylor, J. Biol. Chem. 1989; 264:8443). Isozymesof PKA are found with two types of R subunits (RI and RII), and eachtype has α and β isoforms (Scott, Pharmacol. Ther. 1991; 50:123). Thus,the four isoforms of PKA regulatory subunits are RIα, RIβ, RIIα andRIIβ. The R subunits have been isolated only as stable dimers and thedimerization domain has been shown to consist of the first 44amino-terminal residues of RIIα (Newlon et al., Nat. Struct. Biol. 1999;6:222). As discussed below, similar portions of the amino acid sequencesof other regulatory subunits are involved in dimerization and docking,each located near the N-terminal end of the regulatory subunit. Bindingof cAMP to the R subunits leads to the release of active catalyticsubunits for a broad spectrum of serine/threonine kinase activities,which are oriented toward selected substrates through thecompartmentalization of PKA via its docking with AKAPs (Scott et al., J.Biol. Chem. 1990; 265; 21561)

Since the first AKAP, microtubule-associated protein-2, wascharacterized in 1984 (Lohmann et al., Proc. Natl. Acad. Sci USA. 1984;81:6723), more than 50 AKAPs that localize to various sub-cellularsites, including plasma membrane, actin cytoskeleton, nucleus,mitochondria, and endoplasmic reticulum, have been identified withdiverse structures in species ranging from yeast to humans (Wong andScott, Nat. Rev. Mol. Cell Biol. 2004; 5:959). The AD of AKAPs for PKAis an amphipathic helix of 14-18 residues (Carr et al., J. Biol. Chem.1991; 266:14188). The amino acid sequences of the AD are quite variedamong individual AKAPs, with the binding affinities reported for RIIdimers ranging from 2 to 90 nM (Alto et al., Proc. Natl. Acad. Sci. USA.2003; 100:4445). AKAPs will only bind to dimeric R subunits. For humanRIIcc, the AD binds to a hydrophobic surface formed by the 23amino-terminal residues (Colledge and Scott, Trends Cell Biol. 1999;6:216). Thus, the dimerization domain and AKAP binding domain of humanRIIα are both located within the same N-terminal 44 amino acid sequence(Newlon et al., Nat. Struct. Biol. 1999; 6:222; Newlon et al., EMBO J.2001; 20:1651), which is termed the DDD herein.

We have developed a platform technology to utilize the DDD of human PKAregulatory subunits and the AD of AKAP as an excellent pair of linkermodules for docking any two entities, referred to hereafter as A and B,into a noncovalent complex, which could be further locked into a DNL®complex through the introduction of cysteine residues into both the DDDand AD at strategic positions to facilitate the formation of disulfidebonds. The general methodology of the approach is as follows. Entity Ais constructed by linking a DDD sequence to a precursor of A, resultingin a first component hereafter referred to as a. Because the DDDsequence would effect the spontaneous formation of a dimer, A would thusbe composed of a₂. Entity B is constructed by linking an AD sequence toa precursor of B, resulting in a second component hereafter referred toas b. The dimeric motif of DDD contained in a₂ will create a dockingsite for binding to the AD sequence contained in b, thus facilitating aready association of a₂ and b to form a binary, trimeric complexcomposed of a₂b. This binding event is made irreversible with asubsequent reaction to covalently secure the two entities via disulfidebridges, which occurs very efficiently based on the principle ofeffective local concentration because the initial binding interactionsshould bring the reactive thiol groups placed onto both the DDD and ADinto proximity (Chmura et al., Proc. Natl. Acad. Sci. USA. 2001;98:8480) to ligate site-specifically. Using various combinations oflinkers, adaptor modules and precursors, a wide variety of DNL®constructs of different stoichiometry may be produced and used (see,e.g., U.S. Pat. Nos. 7,550,143; 7,521,056; 7,534,866; 7,527,787 and7,666,400.)

By attaching the DDD and AD away from the functional groups of the twoprecursors, such site-specific ligations are also expected to preservethe original activities of the two precursors. This approach is modularin nature and potentially can be applied to link, site-specifically andcovalently, a wide range of substances, including peptides, proteins,antibodies, antibody fragments, and other effector moieties with a widerange of activities. Utilizing the fusion protein method of constructingAD and DDD conjugated effectors described in the Examples below,virtually any protein or peptide may be incorporated into a DNL®construct. However, the technique is not limiting and other methods ofconjugation may be utilized.

A variety of methods are known for making fusion proteins, includingnucleic acid synthesis, hybridization and/or amplification to produce asynthetic double-stranded nucleic acid encoding a fusion protein ofinterest. Such double-stranded nucleic acids may be inserted intoexpression vectors for fusion protein production by standard molecularbiology techniques (see, e.g. Sambrook et al., Molecular Cloning, Alaboratory manual, 2^(nd) Ed, 1989). In such preferred embodiments, theAD and/or DDD moiety may be attached to either the N-terminal orC-terminal end of an effector protein or peptide. However, the skilledartisan will realize that the site of attachment of an AD or DDD moietyto an effector moiety may vary, depending on the chemical nature of theeffector moiety and the part(s) of the effector moiety involved in itsphysiological activity. Site-specific attachment of a variety ofeffector moieties may be performed using techniques known in the art,such as the use of bivalent cross-linking reagents and/or other chemicalconjugation techniques.

In various embodiments, an antibody or antibody fragment may beincorporated into a DNL® complex by, for example, attaching a DDD or ADmoiety to the C-terminal end of the antibody heavy chain, as describedin detail below. In more preferred embodiments, the DDD or AD moiety,more preferably the AD moiety, may be attached to the C-terminal end ofthe antibody light chain (see, e.g., U.S. patent application Ser. No.13/901,737, filed May 24, 2013, the Examples section of which isincorporated herein by reference.)

Structure-Function Relationships in AD and DDD Moieties

For different types of DNL® constructs, different AD or DDD sequencesmay be utilized. Exemplary DDD and AD sequences are provided below.

DDD1 (SEQ ID NO: 1) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA DDD2(SEQ ID NO: 2) CGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA AD1(SEQ ID NO: 3) QIEYLAKQIVDNAIQQA AD2 (SEQ ID NO: 4)CGQIEYLAKQIVDNAIQQAGC

The skilled artisan will realize that DDD1 and DDD2 are based on the DDDsequence of the human RIIα isoform of protein kinase A. However, inalternative embodiments, the DDD and AD moieties may be based on the DDDsequence of the human RIα form of protein kinase A and a correspondingAKAP sequence, as exemplified in DDD3, DDD3C and AD3 below.

DDD3 (SEQ ID NO: 5) SLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKEEAKDDD3C (SEQ ID NO: 6) MSCGGSLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKEEAK AD3 (SEQ ID NO: 7) CGFEELAWKIAKMIWSDVFQQGC

In other alternative embodiments, other sequence variants of AD and/orDDD moieties may be utilized in construction of the DNL® complexes. Forexample, there are only four variants of human PKA DDD sequences,corresponding to the DDD moieties of PKA RIα, RIIα, RIβ and RIIβ. TheRIIα DDD sequence is the basis of DDD1 and DDD2 disclosed above. Thefour human PKA DDD sequences are shown below. The DDD sequencerepresents residues 1-44 of RIIα, 1-44 of RIIβ, 12-61 of RIα and 13-66of RIβ. (Note that the sequence of DDD1 is modified slightly from thehuman PKA RIIα DDD moiety.)

PKA RIα (SEQ ID NO: 8) SLRECELYVQKHNIQALLKDVSIVQLCTARPERPMAFLREYFEKLEKEEAK PKA RIβ (SEQ ID NO: 9)SLKGCELYVQLHGIQQVLKDCIVHLCISKPERPMKFLREHFEKLEKEENR QILA PKA RIIα(SEQ ID NO: 10) SHIQIPPGLTELLQGYTVEVGQQPPDLVDFAVEYFTRLREARRQ PKA RIIβ(SEQ ID NO: 11) SIEIPAGLTELLQGFTVEVLRHQPADLLEFALQHFTRLQQENER

The structure-function relationships of the AD and DDD domains have beenthe subject of investigation. (See, e.g., Burns-Hamuro et al., 2005,Protein Sci 14:2982-92; Carr et al., 2001, J Biol Chem 276:17332-38;Alto et al., 2003, Proc Natl Acad Sci USA 100:4445-50; Hundsrucker etal., 2006, Biochem J 396:297-306; Stokka et al., 2006, Biochem J400:493-99; Gold et al., 2006, Mol Cell 24:383-95; Kinderman et al.,2006, Mol Cell 24:397-408, the entire text of each of which isincorporated herein by reference.)

For example, Kinderman et al. (2006, Mol Cell 24:397-408) examined thecrystal structure of the AD-DDD binding interaction and concluded thatthe human DDD sequence contained a number of conserved amino acidresidues that were important in either dimer formation or AKAP binding,underlined in SEQ ID NO:1 below. (See FIG. 1 of Kinderman et al., 2006,incorporated herein by reference.) The skilled artisan will realize thatin designing sequence variants of the DDD sequence, one would desirablyavoid changing any of the underlined residues, while conservative aminoacid substitutions might be made for residues that are less critical fordimerization and AKAP binding.

(SEQ ID NO: 1) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA

As discussed in more detail below, conservative amino acid substitutionshave been characterized for each of the twenty common L-amino acids.Thus, based on the data of Kinderman (2006) and conservative amino acidsubstitutions, potential alternative DDD sequences based on SEQ ID NO:1are shown in Table 2. In devising Table 2, only highly conservativeamino acid substitutions were considered. For example, charged residueswere only substituted for residues of the same charge, residues withsmall side chains were substituted with residues of similar size,hydroxyl side chains were only substituted with other hydroxyls, etc.Because of the unique effect of proline on amino acid secondarystructure, no other residues were substituted for proline. A limitednumber of such potential alternative DDD moiety sequences are shown inSEQ ID NO:12 to SEQ ID NO:31 below. The skilled artisan will realizethat an almost unlimited number of alternative species within the genusof DDD moieties can be constructed by standard techniques, for exampleusing a commercial peptide synthesizer or well known site-directedmutagenesis techniques. The effect of the amino acid substitutions on ADmoiety binding may also be readily determined by standard bindingassays, for example as disclosed in Alto et al. (2003, Proc Natl AcadSci USA 100:4445-50).

TABLE 2Conservative Amino Acid Substitutions in DDD1 (SEQ ID NO: 1). Consensussequence disclosed as SEQ ID NO: 87. S H I Q I P P G L T E L L Q G Y T VE V L R T K N A S D N A S D K R Q Q P P D L V E F A V E Y F T R L R E AR A N N E D L D S K K D L K L I I I V V V

(SEQ ID NO: 12) THIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO: 13) SKIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO: 14) SRIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO: 15) SHINIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO: 16) SHIQIPPALTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO: 17) SHIQIPPGLSELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO: 18) SHIQIPPGLTDLLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO: 19) SHIQIPPGLTELLNGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO: 20) SHIQIPPGLTELLQAYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO: 21) SHIQIPPGLTELLQGYSVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO: 22) SHIQIPPGLTELLQGYTVDVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO: 23) SHIQIPPGLTELLQGYTVEVLKQQPPDLVEFAVEYFTRLREARA(SEQ ID NO: 24) SHIQIPPGLTELLQGYTVEVLRNQPPDLVEFAVEYFTRLREARA(SEQ ID NO: 25) SHIQIPPGLTELLQGYTVEVLRQNPPDLVEFAVEYFTRLREARA(SEQ ID NO: 26) SHIQIPPGLTELLQGYTVEVLRQQPPELVEFAVEYFTRLREARA(SEQ ID NO: 27) SHIQIPPGLTELLQGYTVEVLRQQPPDLVDFAVEYFTRLREARA(SEQ ID NO: 28) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFLVEYFTRLREARA(SEQ ID NO: 29) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFIVEYFTRLREARA(SEQ ID NO: 30) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFVVEYFTRLREARA(SEQ ID NO: 31) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVDYFTRLREARA

Alto et al. (2003, Proc Natl Acad Sci USA 100:4445-50) performed abioinformatic analysis of the AD sequence of various AKAP proteins todesign an Rh selective AD sequence called AKAP-IS (SEQ ID NO:3), with abinding constant for DDD of 0.4 nM. The AKAP-IS sequence was designed asa peptide antagonist of AKAP binding to PKA. Residues in the AKAP-ISsequence where substitutions tended to decrease binding to DDD areunderlined in SEQ ID NO:3 below. The skilled artisan will realize thatin designing sequence variants of the AD sequence, one would desirablyavoid changing any of the underlined residues, while conservative aminoacid substitutions might be made for residues that are less critical forDDD binding. Table 3 shows potential conservative amino acidsubstitutions in the sequence of AKAP-IS (AD1, SEQ ID NO:3), similar tothat shown for DDD1 (SEQ ID NO:1) in Table 2 above.

A limited number of such potential alternative AD moiety sequences areshown in SEQ ID NO:32 to SEQ ID NO:49 below. Again, a very large numberof species within the genus of possible AD moiety sequences could bemade, tested and used by the skilled artisan, based on the data of Altoet al. (2003). It is noted that FIG. 2 of Alto (2003) shows an evenlarge number of potential amino acid substitutions that may be made,while retaining binding activity to DDD moieties, based on actualbinding experiments.

AKAP-IS (SEQ ID NO: 3) QIEYLAKQIVDNAIQQA

TABLE 3 Conservative Amino Acid Substitutions in AD1 (SEQ IDNO: 3). Consensus sequence disclosed as SEQ ID NO: 88. Q I E Y L A K Q IV D N A I Q Q A N L D F I R N E Q N N L V T V I S V

(SEQ ID NO: 32) NIEYLAKQIVDNAIQQA (SEQ ID NO: 33) QLEYLAKQIVDNAIQQA(SEQ ID NO: 34) QVEYLAKQIVDNAIQQA (SEQ ID NO: 35) QIDYLAKQIVDNAIQQA(SEQ ID NO: 36) QIEFLAKQIVDNAIQQA (SEQ ID NO: 37) QIETLAKQIVDNAIQQA(SEQ ID NO: 38) QIESLAKQIVDNAIQQA (SEQ ID NO: 39) QIEYIAKQIVDNAIQQA(SEQ ID NO: 40) QIEYVAKQIVDNAIQQA (SEQ ID NO: 41) QIEYLARQIVDNAIQQA(SEQ ID NO: 42) QIEYLAKNIVDNAIQQA (SEQ ID NO: 43) QIEYLAKQIVENAIQQA(SEQ ID NO: 44) QIEYLAKQIVDQAIQQA (SEQ ID NO: 45) QIEYLAKQIVDNAINQA(SEQ ID NO: 46) QIEYLAKQIVDNAIQNA (SEQ ID NO: 47) QIEYLAKQIVDNAIQQL(SEQ ID NO: 48) QIEYLAKQIVDNAIQQI (SEQ ID NO: 49) QIEYLAKQIVDNAIQQV

Gold et al. (2006, Mol Cell 24:383-95) utilized crystallography andpeptide screening to develop a SuperAKAP-IS sequence (SEQ ID NO:50),exhibiting a five order of magnitude higher selectivity for the RHisoform of PKA compared with the RI isoform. Underlined residuesindicate the positions of amino acid substitutions, relative to theAKAP-IS sequence, which increased binding to the DDD moiety of RIIα. Inthis sequence, the N-terminal Q residue is numbered as residue number 4and the C-terminal A residue is residue number 20. Residues wheresubstitutions could be made to affect the affinity for RIIα wereresidues 8, 11, 15, 16, 18, 19 and 20 (Gold et al., 2006). It iscontemplated that in certain alternative embodiments, the SuperAKAP-ISsequence may be substituted for the AKAP-IS AD moiety sequence toprepare DNL® constructs. Other alternative sequences that might besubstituted for the AKAP-IS AD sequence are shown in SEQ ID NO:51-53.Substitutions relative to the AKAP-IS sequence are underlined. It isanticipated that, as with the AD2 sequence shown in SEQ ID NO:4, the ADmoiety may also include the additional N-terminal residues cysteine andglycine and C-terminal residues glycine and cysteine.

SuperAKAP-IS (SEQ ID NO: 50) QIEYVAKQIVDYAIHQAAlternative AKAP sequences (SEQ ID NO: 51) QIEYKAKQIVDHAIHQA(SEQ ID NO: 52) QIEYHAKQIVDHAIHQA (SEQ ID NO: 53) QIEYVAKQIVDHAIHQA

FIG. 2 of Gold et al. disclosed additional DDD-binding sequences from avariety of AKAP proteins, shown below.

RII-Specific AKAPs AKAP-KL (SEQ ID NO: 54) PLEYQAGLLVQNAIQQAI AKAP79(SEQ ID NO: 55) LLIETASSLVKNAIQLSI AKAP-Lbc (SEQ ID NO: 56)LIEEAASRIVDAVIEQVK RI-Specific AKAPs AKAPce (SEQ ID NO: 57)ALYQFADRFSELVISEAL RIAD (SEQ ID NO: 58) LEQVANQLADQIIKEAT PV38(SEQ ID NO: 59) FEELAWKIAKMIWSDVF Dual-Specificity AKAPs AKAP7(SEQ ID NO: 60) ELVRLSKRLVENAVLKAV MAP2D (SEQ ID NO: 61)TAEEVSARIVQVVTAEAV DAKAP1 (SEQ ID NO: 62) QIKQAAFQLISQVILEAT DAKAP2(SEQ ID NO: 63) LAWKIAKMIVSDVMQQ

Stokka et al. (2006, Biochem J400:493-99) also developed peptidecompetitors of AKAP binding to PKA, shown in SEQ ID NO:64-66. Thepeptide antagonists were designated as Ht31 (SEQ ID NO:64), RIAD (SEQ IDNO:65) and PV-38 (SEQ ID NO:66). The Ht-31 peptide exhibited a greateraffinity for the RII isoform of PKA, while the RIAD and PV-38 showedhigher affinity for RI.

Ht31 (SEQ ID NO: 64) DLIEEAASRIVDAVIEQVKAAGAY RIAD (SEQ ID NO: 65)LEQYANQLADQIIKEATE PV-38 (SEQ ID NO: 66) FEELAWKIAKMIWSDVFQQC

Hundsrucker et al. (2006, Biochem J396:297-306) developed still otherpeptide competitors for AKAP binding to PKA, with a binding constant aslow as 0.4 nM to the DDD of the RII form of PKA. The sequences ofvarious AKAP antagonistic peptides are provided in Table 1 ofHundsrucker et al., reproduced in Table 4 below. AKAPIS represents asynthetic RII subunit-binding peptide. All other peptides are derivedfrom the RII-binding domains of the indicated AKAPs.

TABLE 4 AKAP Peptide sequences Peptide Sequence AKAPISQIEYLAKQIVDNAIQQA (SEQ ID NO: 3) AKAPIS-PQIEYLAKQIPDNAIQQA (SEQ ID NO: 67) Ht31KGADLIEEAASRIVDAVIEQVKAAG (SEQ ID NO: 68) Ht31-PKGADLIEEAASRIPDAPIEQVKAAG (SEQ ID NO: 69) AKAP7δ-wt-pepPEDAELVRLSKRLVENAVLKAVQQY (SEQ ID NO: 70) AKAP7δ-L304T-pepPEDAELVRTSKRLVENAVLKAVQQY (SEQ ID NO: 71) AKAP7δ-L308D-pepPEDAELVRLSKRDVENAVLKAVQQY (SEQ ID NO: 72) AKAP7δ-P-pepPEDAELVRLSKRLPENAVLKAVQQY (SEQ ID NO: 73) AKAP7δ-PP-pepPEDAELVRLSKRLPENAPLKAVQQY (SEQ ID NO: 74) AKAP7δ-L314E-pepPEDAELVRLSKRLVENAVEKAVQQY (SEQ ID NO: 75) AKAP1-pepEEGLDRNEEIKRAAFQIISQVISEA (SEQ ID NO: 76) AKAP2-pepLVDDPLEYQAGLLVQNAIQQAIAEQ (SEQ ID NO: 77) AKAP5-pepQYETLLIETASSLVKNAIQLSIEQL (SEQ ID NO: 78) AKAP9-pepLEKQYQEQLEEEVAKVIVSMSIAFA (SEQ ID NO: 79) AKAP10-pepNTDEAQEELAWKIAKMIVSDIMQQA (SEQ ID NO: 80) AKAP11-pepVNLDKKAVLAEKIVAEAIEKAEREL (SEQ ID NO: 81) AKAP12-pepNGILELETKSSKLVQNIIQTAVDQF (SEQ ID NO: 82) AKAP14-pepTQDKNYEDELTQVALALVEDVINYA (SEQ ID NO: 83) Rab32-pepETSAKDNINIEEAARFLVEKILVNH (SEQ ID NO: 84)

Residues that were highly conserved among the AD domains of differentAKAP proteins are indicated below by underlining with reference to theAKAP IS sequence (SEQ ID NO:3). The residues are the same as observed byAlto et al. (2003), with the addition of the C-terminal alanine residue.(See FIG. 4 of Hundsrucker et al. (2006), incorporated herein byreference.) The sequences of peptide antagonists with particularly highaffinities for the RII DDD sequence were those of AKAP-IS,AKAP7δ-wt-pep, AKAP7δ-L304T-pep and AKAP7δ-L308D-pep.

AKAP-IS (SEQ ID NO: 3) QIEYLAKQIVDNAIQQA

Carr et al. (2001, J Biol Chem 276:17332-38) examined the degree ofsequence homology between different AKAP-binding DDD sequences fromhuman and non-human proteins and identified residues in the DDDsequences that appeared to be the most highly conserved among differentDDD moieties. These are indicated below by underlining with reference tothe human PKA RIIα DDD sequence of SEQ ID NO:1. Residues that wereparticularly conserved are further indicated by italics. The residuesoverlap with, but are not identical to those suggested by Kinderman etal. (2006) to be important for binding to AKAP proteins. The skilledartisan will realize that in designing sequence variants of DDD, itwould be most preferred to avoid changing the most conserved residues(italicized), and it would be preferred to also avoid changing theconserved residues (underlined), while conservative amino acidsubstitutions may be considered for residues that are neither underlinednor italicized.

(SEQ ID NO: 1) SHIQ IP P GL TELLQGYT V EVLR Q QP P DLVEFA VE YF TR L REAR A

A modified set of conservative amino acid substitutions for the DDD1(SEQ ID NO:1) sequence, based on the data of Carr et al. (2001) is shownin Table 5. Even with this reduced set of substituted sequences, thereare numerous possible alternative DDD moiety sequences that may beproduced, tested and used by the skilled artisan without undueexperimentation. The skilled artisan could readily derive suchalternative DDD amino acid sequences as disclosed above for Table 2 andTable 3.

TABLE 5Conservative Amino Acid Substitutions in DDD1 (SEQ ID NO: 1). Consensussequence disclosed as SEQ ID NO: 89. S H I Q I P P G L T E L L Q G Y T VE V L R T N S I L A Q Q P P D L V E F A V E Y F T R L R E A R A N L D SK K L L I I I V V V

The skilled artisan will realize that these and other amino acidsubstitutions in the DDD or AD amino acid sequences may be utilized toproduce alternative species within the genus of AD or DDD moieties,using techniques that are standard in the field and only routineexperimentation.

Antibody Allotypes

Immunogenicity of therapeutic antibodies is associated with increasedrisk of infusion reactions and decreased duration of therapeuticresponse (Baert et al., 2003, N Engl J Med 348:602-08). The extent towhich therapeutic antibodies induce an immune response in the host maybe determined in part by the allotype of the antibody (Stickler et al.,2011, Genes and Immunity 12:213-21). Antibody allotype is related toamino acid sequence variations at specific locations in the constantregion sequences of the antibody. The allotypes of IgG antibodiescontaining a heavy chain γ-type constant region are designated as Gmallotypes (1976, J Immunol 117:1056-59).

For the common IgG1 human antibodies, the most prevalent allotype isG1m1 (Stickler et al., 2011, Genes and Immunity 12:213-21). However, theG1m3 allotype also occurs frequently in Caucasians (Id.). It has beenreported that G1m1 antibodies contain allotypic sequences that tend toinduce an immune response when administered to non-G1m1 (nG1m1)recipients, such as G1m3 patients (Id.). Non-G1m1 allotype antibodiesare not as immunogenic when administered to G1m1 patients (Id.).

The human G1m1 allotype comprises the amino acids aspartic acid at Kabatposition 356 and leucine at Kabat position 358 in the CH3 sequence ofthe heavy chain IgG1. The nG1m1 allotype comprises the amino acidsglutamic acid at Kabat position 356 and methionine at Kabat position358. Both G1m1 and nG1m1 allotypes comprise a glutamic acid residue atKabat position 357 and the allotypes are sometimes referred to as DELand EEM allotypes. A non-limiting example of the heavy chain constantregion sequences for G1m1 and nG1m1 allotype antibodies is shown for theexemplary antibodies rituximab (SEQ ID NO:85) and veltuzumab (SEQ IDNO:86).

Rituximab heavy chain constant region sequence (SEQ ID NO: 85)ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKVeltuzumab heavy chain constant region sequence (SEQ ID NO: 86ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Jefferis and Lefranc (2009, mAbs 1:1-7) reviewed sequence variationscharacteristic of IgG allotypes and their effect on immunogenicity. Theyreported that the G1m3 allotype is characterized by an arginine residueat Kabat position 214, compared to a lysine residue at Kabat 214 in theG1m17 allotype. The nG1m1,2 allotype was characterized by glutamic acidat Kabat position 356, methionine at Kabat position 358 and alanine atKabat position 431. The G1m1,2 allotype was characterized by asparticacid at Kabat position 356, leucine at Kabat position 358 and glycine atKabat position 431. In addition to heavy chain constant region sequencevariants, Jefferis and Lefranc (2009) reported allotypic variants in thekappa light chain constant region, with the Km1 allotype characterizedby valine at Kabat position 153 and leucine at Kabat position 191, theKm1,2 allotype by alanine at Kabat position 153 and leucine at Kabatposition 191, and the Km3 allotype characterized by alanine at Kabatposition 153 and valine at Kabat position 191.

With regard to therapeutic antibodies, veltuzumab and rituximab are,respectively, humanized and chimeric IgG1 antibodies against CD20, ofuse for therapy of a wide variety of hematological malignancies. Table 6compares the allotype sequences of rituximab vs. veltuzumab. As shown inTable 6, rituximab (G1m17,1) is a DEL allotype IgG1, with an additionalsequence variation at Kabat position 214 (heavy chain CH1) of lysine inrituximab vs. arginine in veltuzumab. It has been reported thatveltuzumab is less immunogenic in subjects than rituximab (see, e.g.,Morchhauser et al., 2009, J Clin Oncol 27:3346-53; Goldenberg et al.,2009, Blood 113:1062-70; Robak & Robak, 2011, BioDrugs 25:13-25), aneffect that has been attributed to the difference between humanized andchimeric antibodies. However, the difference in allotypes between theEEM and DEL allotypes likely also accounts for the lower immunogenicityof veltuzumab.

TABLE 6 Allotypes of Rituximab vs. Veltuzumab Heavy chain position andassociated allotypes Complete 214 356/358 431 allotype (allotype)(allotype) (allotype) Rituximab G1m17, 1 K 17 D/L 1 A — Veltuzumab G1m3R 3 E/M — A —

In order to reduce the immunogenicity of therapeutic antibodies inindividuals of nG1m1 genotype, it is desirable to select the allotype ofthe antibody to correspond to the G1m3 allotype, characterized byarginine at Kabat 214, and the nG1m1,2 null-allotype, characterized byglutamic acid at Kabat position 356, methionine at Kabat position 358and alanine at Kabat position 431. Surprisingly, it was found thatrepeated subcutaneous administration of G1m3 antibodies over a longperiod of time did not result in a significant immune response. Inalternative embodiments, the human IgG4 heavy chain in common with theG1m3 allotype has arginine at Kabat 214, glutamic acid at Kabat 356,methionine at Kabat 359 and alanine at Kabat 431. Since immunogenicityappears to relate at least in part to the residues at those locations,use of the human IgG4 heavy chain constant region sequence fortherapeutic antibodies is also a preferred embodiment. Combinations ofG1m3 IgG1 antibodies with IgG4 antibodies may also be of use fortherapeutic administration.

Amino Acid Substitutions

In alternative embodiments, the disclosed methods and compositions mayinvolve production and use of proteins or peptides with one or moresubstituted amino acid residues. For example, the DDD and/or ADsequences used to make DNL® constructs may be modified as discussedabove.

The skilled artisan will be aware that, in general, amino acidsubstitutions typically involve the replacement of an amino acid withanother amino acid of relatively similar properties (i.e., conservativeamino acid substitutions). The properties of the various amino acids andeffect of amino acid substitution on protein structure and function havebeen the subject of extensive study and knowledge in the art.

For example, the hydropathic index of amino acids may be considered(Kyte & Doolittle, 1982, J. Mol. Biol., 157:105-132). The relativehydropathic character of the amino acid contributes to the secondarystructure of the resultant protein, which in turn defines theinteraction of the protein with other molecules. Each amino acid hasbeen assigned a hydropathic index on the basis of its hydrophobicity andcharge characteristics (Kyte & Doolittle, 1982), these are: isoleucine(+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine(−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine(−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine(−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine(−4.5). In making conservative substitutions, the use of amino acidswhose hydropathic indices are within ±2 is preferred, within ±1 are morepreferred, and within ±0.5 are even more preferred.

Amino acid substitution may also take into account the hydrophilicity ofthe amino acid residue (e.g., U.S. Pat. No. 4,554,101). Hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0); glutamate (+3.0); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (O); threonine (−0.4);proline (−0.5.+−0.1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). Replacement ofamino acids with others of similar hydrophilicity is preferred.

Other considerations include the size of the amino acid side chain. Forexample, it would generally not be preferred to replace an amino acidwith a compact side chain, such as glycine or serine, with an amino acidwith a bulky side chain, e.g., tryptophan or tyrosine. The effect ofvarious amino acid residues on protein secondary structure is also aconsideration. Through empirical study, the effect of different aminoacid residues on the tendency of protein domains to adopt analpha-helical, beta-sheet or reverse turn secondary structure has beendetermined and is known in the art (see, e.g., Chou & Fasman, 1974,Biochemistry, 13:222-245; 1978, Ann. Rev. Biochem., 47: 251-276; 1979,Biophys. J., 26:367-384).

Based on such considerations and extensive empirical study, tables ofconservative amino acid substitutions have been constructed and areknown in the art. For example: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine. Alternatively: Ala (A) leu, ile, val; Arg (R)gln, asn, lys; Asn (N) his, asp, lys, arg, gln; Asp (D) asn, glu; Cys(C) ala, ser; Gln (Q) glu, asn; Glu (E) gln, asp; Gly (G) ala; His (H)asn, gln, lys, arg; Ile (I) val, met, ala, phe, leu; Leu (L) val, met,ala, phe, ile; Lys (K) gln, asn, arg; Met (M) phe, ile, leu; Phe (F)leu, val, ile, ala, tyr; Pro (P) ala; Ser (S), thr; Thr (T) ser; Trp (W)phe, tyr; Tyr (Y) trp, phe, thr, ser; Val (V) ile, leu, met, phe, ala.

Other considerations for amino acid substitutions include whether or notthe residue is located in the interior of a protein or is solventexposed. For interior residues, conservative substitutions wouldinclude: Asp and Asn; Ser and Thr; Ser and Ala; Thr and Ala; Ala andGly; Ile and Val; Val and Leu; Leu and Ile; Leu and Met; Phe and Tyr;Tyr and Trp. (See, e.g., PROWL website at rockefeller.edu) For solventexposed residues, conservative substitutions would include: Asp and Asn;Asp and Glu; Glu and Gln; Glu and Ala; Gly and Asn; Ala and Pro; Ala andGly; Ala and Ser; Ala and Lys; Ser and Thr; Lys and Arg; Val and Leu;Leu and Ile; Ile and Val; Phe and Tyr. (Id.) Various matrices have beenconstructed to assist in selection of amino acid substitutions, such asthe PAM250 scoring matrix, Dayhoff matrix, Grantham matrix, McLachlanmatrix, Doolittle matrix, Henikoff matrix, Miyata matrix, Fitch matrix,Jones matrix, Rao matrix, Levin matrix and Risler matrix (Idem.)

In determining amino acid substitutions, one may also consider theexistence of intermolecular or intramolecular bonds, such as formationof ionic bonds (salt bridges) between positively charged residues (e.g.,His, Arg, Lys) and negatively charged residues (e.g., Asp, Glu) ordisulfide bonds between nearby cysteine residues.

Methods of substituting any amino acid for any other amino acid in anencoded protein sequence are well known and a matter of routineexperimentation for the skilled artisan, for example by the technique ofsite-directed mutagenesis or by synthesis and assembly ofoligonucleotides encoding an amino acid substitution and splicing intoan expression vector construct.

Avimers

In certain embodiments, the binding moieties described herein maycomprise one or more avimer sequences. Avimers are a class of bindingproteins somewhat similar to antibodies in their affinities andspecificities for various target molecules. They were developed fromhuman extracellular receptor domains by in vitro exon shuffling andphage display. (Silverman et al., 2005, Nat. Biotechnol. 23:1493-94;Silverman et al., 2006, Nat. Biotechnol. 24:220). The resultingmultidomain proteins may comprise multiple independent binding domains,that may exhibit improved affinity (in some cases sub-nanomolar) andspecificity compared with single-epitope binding proteins. (Id.) Invarious embodiments, avimers may be attached to, for example, DDD and/orAD sequences for use in the claimed methods and compositions. Additionaldetails concerning methods of construction and use of avimers aredisclosed, for example, in U.S. Patent Application Publication Nos.20040175756, 20050048512, 20050053973, 20050089932 and 20050221384, theExamples section of each of which is incorporated herein by reference.

Phage Display

Certain embodiments of the claimed compositions and/or methods mayconcern binding peptides and/or peptide mimetics of various targetmolecules, cells or tissues. Binding peptides may be identified by anymethod known in the art, including but not limiting to the phage displaytechnique. Various methods of phage display and techniques for producingdiverse populations of peptides are well known in the art. For example,U.S. Pat. Nos. 5,223,409; 5,622,699 and 6,068,829 disclose methods forpreparing a phage library. The phage display technique involvesgenetically manipulating bacteriophage so that small peptides can beexpressed on their surface (Smith and Scott, 1985, Science228:1315-1317; Smith and Scott, 1993, Meth. Enzymol. 21:228-257). Inaddition to peptides, larger protein domains such as single-chainantibodies may also be displayed on the surface of phage particles (Arapet al., 1998, Science 279:377-380).

Targeting amino acid sequences selective for a given organ, tissue, celltype or target molecule may be isolated by panning (Pasqualini andRuoslahti, 1996, Nature 380:364-366; Pasqualini, 1999, The Quart. J.Nucl. Med. 43:159-162). In brief, a library of phage containing putativetargeting peptides is administered to an intact organism or to isolatedorgans, tissues, cell types or target molecules and samples containingbound phage are collected. Phage that bind to a target may be elutedfrom a target organ, tissue, cell type or target molecule and thenamplified by growing them in host bacteria.

In certain embodiments, the phage may be propagated in host bacteriabetween rounds of panning. Rather than being lysed by the phage, thebacteria may instead secrete multiple copies of phage that display aparticular insert. If desired, the amplified phage may be exposed to thetarget organs, tissues, cell types or target molecule again andcollected for additional rounds of panning. Multiple rounds of panningmay be performed until a population of selective or specific binders isobtained. The amino acid sequence of the peptides may be determined bysequencing the DNA corresponding to the targeting peptide insert in thephage genome. The identified targeting peptide may then be produced as asynthetic peptide by standard protein chemistry techniques (Arap et al.,1998, Smith et al., 1985).

In some embodiments, a subtraction protocol may be used to furtherreduce background phage binding. The purpose of subtraction is to removephage from the library that bind to targets other than the target ofinterest. In alternative embodiments, the phage library may beprescreened against a control cell, tissue or organ. For example,tumor-binding peptides may be identified after prescreening a libraryagainst a control normal cell line. After subtraction the library may bescreened against the molecule, cell, tissue or organ of interest. Othermethods of subtraction protocols are known and may be used in thepractice of the claimed methods, for example as disclosed in U.S. Pat.Nos. 5,840,841, 5,705,610, 5,670,312 and 5,492,807.

Aptamers

In certain embodiments, a targeting moiety of use may be an aptamer.Methods of constructing and determining the binding characteristics ofaptamers are well known in the art. For example, such techniques aredescribed in U.S. Pat. Nos. 5,582,981, 5,595,877 and 5,637,459, theExamples section of each incorporated herein by reference. Methods forpreparation and screening of aptamers that bind to particular targets ofinterest are well known, for example U.S. Pat. Nos. 5,475,096 and5,270,163, the Examples section of each incorporated herein byreference.

Aptamers may be prepared by any known method, including synthetic,recombinant, and purification methods, and may be used alone or incombination with other ligands specific for the same target. In general,a minimum of approximately 3 nucleotides, preferably at least 5nucleotides, are necessary to effect specific binding. Aptamers ofsequences shorter than 10 bases may be feasible, although aptamers of10, 20, 30 or 40 nucleotides may be preferred.

Aptamers may be isolated, sequenced, and/or amplified or synthesized asconventional DNA or RNA molecules. Alternatively, aptamers of interestmay comprise modified oligomers. Any of the hydroxyl groups ordinarilypresent in aptamers may be replaced by phosphonate groups, phosphategroups, protected by a standard protecting group, or activated toprepare additional linkages to other nucleotides, or may be conjugatedto solid supports. One or more phosphodiester linkages may be replacedby alternative linking groups, such as P(O)O replaced by P(O)S, P(O)NR₂,P(O)R, P(O)OR′, CO, or CNR₂, wherein R is H or alkyl (1-20C) and R′ isalkyl (1-20C); in addition, this group may be attached to adjacentnucleotides through O or S. Not all linkages in an oligomer need to beidentical.

Affibodies and Fynomers

Certain alternative embodiments may utilize affibodies in place ofantibodies. Affibodies are commercially available from Affibody AB(Solna, Sweden). Affibodies are small proteins that function as antibodymimetics and are of use in binding target molecules. Affibodies weredeveloped by combinatorial engineering on an alpha helical proteinscaffold (Nord et al., 1995, Protein Eng 8:601-8; Nord et al., 1997, NatBiotechnol 15:772-77). The affibody design is based on a three helixbundle structure comprising the IgG binding domain of protein A (Nord etal., 1995; 1997). Affibodies with a wide range of binding affinities maybe produced by randomization of thirteen amino acids involved in the Fcbinding activity of the bacterial protein A (Nord et al., 1995; 1997).After randomization, the PCR amplified library was cloned into aphagemid vector for screening by phage display of the mutant proteins.The phage display library may be screened against any known antigen,using standard phage display screening techniques (e.g., Pasqualini andRuoslahti, 1996, Nature 380:364-366; Pasqualini, 1999, Quart. J. Nucl.Med. 43:159-162), in order to identify one or more affibodies againstthe target antigen.

A ¹⁷⁷Lu-labeled affibody specific for HER2/neu has been demonstrated totarget HER2-expressing xenografts in vivo (Tolmachev et al., 2007,Cancer Res 67:2773-82). Although renal toxicity due to accumulation ofthe low molecular weight radiolabeled compound was initially a problem,reversible binding to albumin reduced renal accumulation, enablingradionuclide-based therapy with labeled affibody (Id.).

The feasibility of using radiolabeled affibodies for in vivo tumorimaging has been recently demonstrated (Tolmachev et al., 2011,Bioconjugate Chem 22:894-902). A maleimide-derivatized NOTA wasconjugated to the anti-HER2 affibody and radiolabeled with ¹¹¹In (Id.).Administration to mice bearing the HER2-expressing DU-145 xenograft,followed by gamma camera imaging, allowed visualization of the xenograft(Id.).

Fynomers can also bind to target antigens with a similar affinity andspecificity to antibodies. Fynomers are based on the human Fyn SH3domain as a scaffold for assembly of binding molecules. The Fyn SH3domain is a fully human, 63 amino acid protein that can be produced inbacteria with high yields. Fynomers may be linked together to yield amultispecific binding protein with affinities for two or more differentantigen targets. Fynomers are commercially available from COVAGEN AG(Zurich, Switzerland).

The skilled artisan will realize that affibodies or fynomers may be usedas targeting molecules in the practice of the claimed methods andcompositions.

Conjugation Protocols

The preferred conjugation protocol is based on a thiol-maleimide, athiol-vinylsulfone, a thiol-bromoacetamide, or a thiol-iodoacetamidereaction that is facile at neutral or acidic pH. This obviates the needfor higher pH conditions for conjugations as, for instance, would benecessitated when using active esters. Further details of exemplaryconjugation protocols are described below in the Examples section.

Therapeutic Treatment

In another aspect, the invention relates to a method of treating asubject, comprising administering a therapeutically effective amount ofan antibody or immunoconjugate as described herein to a subject,preferably in combination with a PARP inhibitor, microtubule inhibitor,Bruton kinase inhibitor and/or PI3K inhibitor. Diseases that may betreated with the antibodies or immunoconjugates described hereininclude, but are not limited to B-cell malignancies (e.g., non-Hodgkin'slymphoma, mantle cell lymphoma, multiple myeloma, Hodgkin's lymphoma,diffuse large B cell lymphoma, Burkitt lymphoma, follicular lymphoma,acute lymphocytic leukemia, chronic lymphocytic leukemia, hairy cellleukemia) using, for example an anti-CD22 antibody such as the hLL2 MAb(epratuzumab, see U.S. Pat. No. 6,183,744), against another CD22 epitope(hRFB4) or antibodies against other B-cell antigens, such as CD19, CD20,CD21, CD22, CD23, CD37, CD40, CD40L, CD52, CD74, CD80 or HLA-DR. Otherdiseases include, but are not limited to, adenocarcinomas ofendodermally-derived digestive system epithelia, cancers such as breastcancer and non-small cell lung cancer, and other carcinomas, sarcomas,glial tumors, myeloid leukemias, etc. In particular, antibodies againstan antigen, e.g., an oncofetal antigen, produced by or associated with amalignant solid tumor or hematopoietic neoplasm, e.g., agastrointestinal, stomach, colon, esophageal, liver, lung, breast,pancreatic, liver, prostate, ovarian, testicular, brain, bone orlymphatic tumor, a sarcoma or a melanoma, are advantageously used. Suchtherapeutics can be given once or repeatedly, depending on the diseasestate and tolerability of the conjugate, and can also be used optionallyin combination with other therapeutic modalities, such as surgery,external radiation, radioimmunotherapy, immunotherapy, chemotherapy,antisense therapy, interference RNA therapy, gene therapy, and the like.Each combination will be adapted to the tumor type, stage, patientcondition and prior therapy, and other factors considered by themanaging physician.

As used herein, the term “subject” refers to any animal (i.e.,vertebrates and invertebrates) including, but not limited to mammals,including humans. It is not intended that the term be limited to aparticular age or sex. Thus, adult and newborn subjects, as well asfetuses, whether male or female, are encompassed by the term. Dosesgiven herein are for humans, but can be adjusted to the size of othermammals, as well as children, in accordance with weight or square metersize. Where an antibody or immunoconjugate is administered to a humansubject, the person of ordinary skill will realize that the targetantigen to which the antibody or immunoconjugate binds will be a humanantigen.

In a preferred embodiment, antibodies or immunoconjugates comprising ananti-EGP-1 (anti-Trop-2) antibody, such as the hRS7 Mab, can be used totreat carcinomas such as carcinomas of the esophagus, pancreas, lung,stomach, colon and rectum, urinary bladder, breast, ovary, uterus,kidney and prostate, as disclosed in U.S. Pat. Nos. 7,238,785; 7,517,964and 8,084,583, the Examples section of which is incorporated herein byreference. An hRS7 antibody is a humanized antibody that comprises lightchain complementarity-determining region (CDR) sequences CDR1(KASQDVSIAVA, SEQ ID NO:90); CDR2 (SASYRYT, SEQ ID NO:91); and CDR3(QQHYITPLT, SEQ ID NO:92) and heavy chain CDR sequences CDR1 (NYGMN, SEQID NO:93); CDR2 (WINTYTGEPTYTDDFKG, SEQ ID NO:94) and CDR3(GGFGSSYWYFDV, SEQ ID NO:95).

In another preferred embodiment, antibodies or immunoconjugatescomprising an anti-CEACAM5 antibody (e.g., hMN-14, labretuzumab) and/oran anti-CEACAM6 antibody (e.g., hMN-3 or hMN-15) may be used to treatany of a variety of cancers that express CEACAM5 and/or CEACAM6, asdisclosed in U.S. Pat. Nos. 7,541,440; 7,951,369; 5,874,540; 6,676,924and 8,267,865, the Examples section of each incorporated herein byreference. Solid tumors that may be treated using anti-CEACAM5,anti-CEACAM6, or a combination of the two, include but are not limitedto breast, lung, pancreatic, esophageal, medullary thyroid, ovarian,colon, rectum, urinary bladder, mouth and stomach cancers. A majority ofcarcinomas, including gastrointestinal, respiratory, genitourinary andbreast cancers express CEACAM5 and may be treated with the subjectantibodies or immunoconjugates. An hMN-14 antibody is a humanizedantibody that comprises light chain variable region CDR sequences CDR1(KASQDVGTSVA; SEQ ID NO:96), CDR2 (WTSTRHT; SEQ ID NO:97), and CDR3(QQYSLYRS; SEQ ID NO:98), and the heavy chain variable region CDRsequences CDR1 (TYWMS; SEQ ID NO:99), CDR2 (EIHPDSSTINYAPSLKD; SEQ IDNO:100) and CDR3 (LYFGFPWFAY; SEQ ID NO:101). An hMN-3 antibody is ahumanized antibody that comprises light chain variable region CDRsequences CDR1 (RSSQSIVHSNGNTYLE, SEQ ID NO:102), CDR2 (KVSNRFS, SEQ IDNO:103) and CDR3 (FQGSHVPPT, SEQ ID NO:104) and the heavy chain CDRsequences CDR1 (NYGMN, SEQ ID NO:105), CDR2 (WINTYTGEPTYADDFKG, SEQ IDNO:106) and CDR3 (KGWMDFNSSLDY, SEQ ID NO:107). An hMN-15 antibody is ahumanized antibody that comprises light chain variable region CDRsequences SASSRVSYIH (SEQ ID NO:108); GTSTLAS (SEQ ID NO:109); andQQWSYNPPT (SEQ ID NO:110); and heavy chain variable region CDR sequencesDYYMS (SEQ ID NO:111); FIANKANGHTTDYSPSVKG (SEQ ID NO:112); andDMGIRWNFDV (SEQ ID NO:113).

In another preferred embodiment, antibodies or immunoconjugatescomprising an anti-CD74 antibody (e.g., hLL1, milatuzumab, disclosed inU.S. Pat. Nos. 7,074,403; 7,312,318; 7,772,373; 7,919,087 and 7,931,903,the Examples section of each incorporated herein by reference) may beused to treat any of a variety of cancers that express CD74, includingbut not limited to renal, lung, intestinal, stomach, breast, prostate orovarian cancer, as well as several hematological cancers, such asmultiple myeloma, chronic lymphocytic leukemia, acute lymphoblasticleukemia, non-Hodgkin lymphoma, and Hodgkin lymphoma. An hLL1 antibodyis a humanized antibody comprising the light chain CDR sequences CDR1(RSSQSLVHRNGNTYLH; SEQ ID NO:114), CDR2 (TVSNRFS; SEQ ID NO:115), andCDR3 (SQSSHVPPT; SEQ ID NO:116) and the heavy chain variable region CDRsequences CDR1 (NYGVN; SEQ ID NO:117), CDR2 (WINPNTGEPTFDDDFKG; SEQ IDNO:118), and CDR3 (SRGKNEAWFAY; SEQ ID NO:119).

In another preferred embodiment, antibodies or immunoconjugatescomprising an anti-CD22 antibody (e.g., hLL2, epratuzumab, disclosed inU.S. Pat. Nos. 5,789,554; 6,183,744; 6,187,287; 6,306,393; 7,074,403 and7,641,901, the Examples section of each incorporated herein byreference, or the chimeric or humanized RFB4 antibody) may be used totreat any of a variety of cancers that express CD22, including but notlimited to indolent forms of B-cell lymphomas, aggressive forms ofB-cell lymphomas, chronic lymphatic leukemias, acute lymphaticleukemias, non-Hodgkin's lymphoma, Hodgkin's lymphoma, Burkitt lymphoma,follicular lymphoma or diffuse B-cell lymphoma. An hLL2 antibody is ahumanized antibody comprising light chain CDR sequences CDR1(KSSQSVLYSANHKYLA, SEQ ID NO:120), CDR2 (WASTRES, SEQ ID NO:121), andCDR3 (HQYLSSWTF, SEQ ID NO:122) and the heavy chain CDR sequences CDR1(SYWLH, SEQ ID NO:123), CDR2 (YINPRNDYTEYNQNFKD, SEQ ID NO:124), andCDR3 (RDITTFY, SEQ ID NO:125)

In a preferred embodiment, antibodies or immunoconjugates comprisinganti-CSAp antibodies, such as the hMu-9 MAb, can be used to treatcolorectal, as well as pancreatic and ovarian cancers as disclosed inU.S. Pat. Nos. 6,962,702; 7,387,772; 7,414,121; 7,553,953; 7,641,891 and7,670,804, the Examples section of each incorporated herein byreference. In addition, antibodies or immunoconjugates comprising thehPAM4 MAb can be used to treat pancreatic cancer or other solid tumors,as disclosed in U.S. Pat. Nos. 7,238,786 and 7,282,567, the Examplessection of each incorporated herein by reference. An hMu-9 antibody is ahumanized antibody comprising light chain CDR sequences CDR1(RSSQSIVHSNGNTYLE, SEQ ID NO:126), CDR2 (KVSNRFS, SEQ ID NO:127), andCDR3 (FQGSRVPYT, SEQ ID NO:128), and heavy chain variable CDR sequencesCDR1 (EYVIT, SEQ ID NO:129), CDR2 (EIYPGSGSTSYNEKFK, SEQ ID NO:130), andCDR3 (EDL, SEQ ID NO:131). An hPAM4 antibody is a humanized antibodycomprising light chain variable region CDR sequences CDR1 (SASSSVSSSYLY,SEQ ID NO:132); CDR2 (STSNLAS, SEQ ID NO:133); and CDR3 (HQWNRYPYT, SEQID NO:134); and heavy chain CDR sequences CDR1 (SYVLH, SEQ ID NO:135);CDR2 (YINPYNDGTQYNEKFKG, SEQ ID NO:136) and CDR3 (GFGGSYGFAY, SEQ IDNO:137).

In another preferred embodiment, antibodies or immunoconjugatescomprising an anti-alpha fetoprotein (AFP) MAb, such as IMMU31, can beused to treat hepatocellular carcinoma, germ cell tumors, and otherAFP-producing tumors using humanized, chimeric and human antibody forms,as disclosed in U.S. Pat. No. 7,300,655, the Examples section of whichis incorporated herein by reference. An IMMU31 antibody is a humanizedantibody comprising the heavy chain CDR sequences CDR1 (SYVIH, SEQ IDNO:138), CDR2 (YIHPYNGGTKYNEKFKG, SEQ ID NO:139) and CDR3 (SGGGDPFAY,SEQ ID NO:140) and the light chain CDR1 (KASQDINKYIG, SEQ ID NO:141),CDR2 (YTSALLP, SEQ ID NO:142) and CDR3 (LQYDDLWT, SEQ ID NO:143).

In another preferred embodiment, antibodies or immunoconjugatescomprising an anti-HLA-DR MAb, such as hL243 (IMMU-114), can be used totreat lymphoma, leukemia, multiple myeloma, cancers of the skin,esophagus, stomach, colon, rectum, pancreas, lung, breast, ovary,bladder, endometrium, cervix, testes, kidney, liver, melanoma or otherHLA-DR-producing tumors, as disclosed in U.S. Pat. No. 7,612,180, theExamples section of which is incorporated herein by reference. An hL243antibody is a humanized antibody comprising the heavy chain CDRsequences CDR1 (NYGMN, SEQ ID NO:144), CDR2 (WINTYTREPTYADDFKG, SEQ IDNO:145), and CDR3 (DITAVVPTGFDY, SEQ ID NO:146) and light chain CDRsequences CDR1 (RASENIYSNLA, SEQ ID NO:147), CDR2 (AASNLAD, SEQ IDNO:148), and CDR3 (QHFWTTPWA, SEQ ID NO:149).

In another preferred embodiment, antibodies or immunoconjugatescomprising an anti-CD20 MAb, such as veltuzumab (hA20), IF5,obinutuzumab (GA101), or rituximab, can be used to treat lymphoma,leukemia, immune thrombocytopenic purpura, systemic lupus erythematosus,Sjögren's syndrome, Evans syndrome, arthritis, arteritis, pemphigusvulgaris, renal graft rejection, cardiac graft rejection, rheumatoidarthritis, Burkitt lymphoma, non-Hodgkin's lymphoma, follicularlymphoma, small lymphocytic lymphoma, diffuse B-cell lymphoma, marginalzone lymphoma, chronic lymphocytic leukemia, acute lymphocytic leukemia,Type I diabetes mellitus, GVHD, or multiple sclerosis, as disclosed inU.S. Pat. No. 7,435,803 or 8,287,864, the Examples section of eachincorporated herein by reference. An hA20 (veltuzumab) antibody is ahumanized antibody comprising the light chain CDR sequences CDRL1(RASSSVSYIH, SEQ ID NO:150), CDRL2 (ATSNLAS, SEQ ID NO:151) and CDRL3(QQWTSNPPT, SEQ ID NO:152) and heavy chain CDR sequences CDRH1 (SYNMH,SEQ ID NO:153), CDRH2 (AIYPGNGDTSYNQKFKG, SEQ ID NO:154) and CDRH3(STYYGGDWYFDV, SEQ ID NO:155).

In another preferred embodiment, antibodies or immunoconjugatescomprising an anti-CD19 MAb, such as hA19, can be used to treat B-cellrelated lymphomas and leukemias, such as non-Hodgkin's lymphoma, chroniclymphocytic leukemia or acute lymphoblastic leukemia. Other diseasestates that may be treated include autoimmune diseases, such as acute orchronic immune thrombocytopenia, dermatomyositis, Sydenham's chorea,myasthenia gravis, systemic lupus erythematosus, lupus nephritis,rheumatic fever, polyglandular syndromes, bullous pemphigoid, diabetesmellitus, Henoch-Schonlein purpura, post-streptococcal nephritis,erythema nodosum, Takayasu's arteritis, Addison's disease, rheumatoidarthritis, multiple sclerosis, sarcoidosis, ulcerative colitis, erythemamultiforme, IgA nephropathy, polyarteritis nodosa, ankylosingspondylitis, Goodpasture's syndrome, thromboangitis ubiterans, Sjögren'ssyndrome, primary biliary cirrhosis, Hashimoto's thyroiditis,thyrotoxicosis, scleroderma, chronic active hepatitis,polymyositis/dermatomyositis, polychondritis, pemphigus vulgaris,Wegener's granulomatosis, membranous nephropathy, amyotrophic lateralsclerosis, tabes dorsalis, giant cell arteritis/polymyalgia, perniciousanemia, rapidly progressive glomerulonephritis, psoriasis, and fibrosingalveolitis, as disclosed in U.S. Pat. Nos. 7,109,304, 7,462,352,7,902,338, 8,147,831 and 8,337,840, the Examples section of eachincorporated herein by reference. An hA19 antibody is a humanizedantibody comprising the light chain CDR sequences CDR1 KASQSVDYDGDSYLN(SEQ ID NO: 156); CDR2 DASNLVS (SEQ ID NO: 157); and CDR3 QQSTEDPWT (SEQID NO: 158) and the heavy chain CDR sequences CDR1 SYWMN (SEQ ID NO:159); CDR2 QIWPGDGDTNYNGKFKG (SEQ ID NO: 160) and CDR3 RETTTVGRYYYAMDY(SEQ ID NO: 161).

In another preferred embodiment, antibodies or immunoconjugatescomprising anti-tenascin antibodies can be used to treat hematopoieticand solid tumors, and conjugates comprising antibodies to tenascin canbe used to treat solid tumors, preferably brain cancers likeglioblastomas.

In a preferred embodiment, the antibodies that are used in the treatmentof human disease are human or humanized (CDR-grafted) versions ofantibodies, although murine and chimeric versions of antibodies can beused. Same species IgG molecules as delivery agents are mostly preferredto minimize immune responses. This is particularly important whenconsidering repeat treatments. For humans, a human or humanized IgGantibody is less likely to generate an anti-IgG immune response frompatients. Antibodies such as hLL1 and hLL2 rapidly internalize afterbinding to internalizing antigen on target cells, which means that thedrug being carried is rapidly internalized into cells as well. However,antibodies that have slower rates of internalization can also be used toeffect selective therapy.

In another preferred embodiment, the antibodies or immunoconjugates canbe used to treat autoimmune disease or immune system dysfunction (e.g.,graft-versus-host disease, organ transplant rejection). Antibodies ofuse to treat autoimmune/immune dysfunction disease may bind to exemplaryantigens including, but not limited to, BCL-1, BCL-2, BCL-6, CD1a, CD2,CD3, CD4, CD5, CD7, CD8, CD10, CD11b, CD11c, CD13, CD14, CD15, CD16,CD19, CD20, CD21, CD22, CD23, CD25, CD33, CD34, CD38, CD40, CD40L,CD41a, CD43, CD45, CD55, CD56, CCD57, CD59, CD64, CD71, CD74, CD79a,CD79b, CD117, CD138, FMC-7 and HLA-DR. Antibodies that bind to these andother target antigens, discussed above, may be used to treat autoimmuneor immune dysfunction diseases. Autoimmune diseases that may be treatedwith antibodies or immunoconjugates may include acute idiopathicthrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura,dermatomyositis, Sydenham's chorea, myasthenia gravis, systemic lupuserythematosus, lupus nephritis, rheumatic fever, polyglandularsyndromes, bullous pemphigoid, diabetes mellitus, Henoch-Schonleinpurpura, post-streptococcal nephritis, erythema nodosum, Takayasu'sarteritis, ANCA-associated vasculitides, Addison's disease, rheumatoidarthritis, multiple sclerosis, sarcoidosis, ulcerative colitis, erythemamultiforme, IgA nephropathy, polyarteritis nodosa, ankylosingspondylitis, Goodpasture's syndrome, thromboangitis obliterans,Sjogren's syndrome, primary biliary cirrhosis, Hashimoto's thyroiditis,thyrotoxicosis, scleroderma, chronic active hepatitis,polymyositis/dermatomyositis, polychondritis, bullous pemphigoid,pemphigus vulgaris, Wegener's granulomatosis, membranous nephropathy,amyotrophic lateral sclerosis, tabes dorsalis, giant cellarteritis/polymyalgia, pernicious anemia, rapidly progressiveglomerulonephritis, psoriasis or fibrosing alveolitis.

In another preferred embodiment, a therapeutic agent used in combinationwith the immunoconjugates of this invention may comprise one or moreisotopes. Radioactive isotopes useful for treating diseased tissueinclude, but are not limited to—¹¹¹In, ¹⁷⁷Lu, ²¹²Bi, ²¹³Bi, ²¹¹At, ⁶²Cu,⁶⁷Cu, ⁹⁰Y, ¹²⁵I, ¹³¹I, ³²P, ³³P, ⁴⁷Sc, ¹¹¹Ag, ⁶⁷Ga, ¹⁴²Pr, ¹⁵³Sm, ¹⁶¹Tb,¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ²¹²Pb, ²²³Ra, ²²⁵Ac, ⁵⁹Fe, ⁷⁵Se,⁷⁷As, ⁸⁹Sr, ⁹⁹Mo, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹⁴³Pr, ¹⁴⁹Pm, ¹⁶⁹Et, ¹⁹⁴Ir, ¹⁹⁸Au,¹⁹⁹Au, ²⁷⁷Th and ²¹¹Pb. The therapeutic radionuclide preferably has adecay-energy in the range of 20 to 6,000 keV, preferably in the ranges60 to 200 keV for an Auger emitter, 100-2,500 keV for a beta emitter,and 4,000-6,000 keV for an alpha emitter. Maximum decay energies ofuseful beta-particle-emitting nuclides are preferably 20-5,000 keV, morepreferably 100-4,000 keV, and most preferably 500-2,500 keV. Alsopreferred are radionuclides that substantially decay with Auger-emittingparticles. For example, Co-58, Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109,In-111, Sb-119, 1-125, Ho-161, Os-189m and Ir-192. Decay energies ofuseful beta-particle-emitting nuclides are preferably <1,000 keV, morepreferably <100 keV, and most preferably <70 keV. Also preferred areradionuclides that substantially decay with generation ofalpha-particles. Such radionuclides include, but are not limited to:Dy-152, At-211, Bi-212, Ra-223, Rn-219, Po-215, Bi-211, Ac-225, Fr-221,At-217, Bi-213, Th-227 and Fm-255. Decay energies of usefulalpha-particle-emitting radionuclides are preferably 2,000-10,000 keV,more preferably 3,000-8,000 keV, and most preferably 4,000-7,000 keV.Additional potential radioisotopes of use include ¹¹C, ¹³N, ¹⁵O, ⁷⁵Br,¹⁹⁸Au, ²²⁴Ac, ¹²⁶I, ¹³³I, ⁷⁷Br, ^(113m)In, ⁹⁵Ru, ⁹⁷Ru, ¹⁰³Ru, ¹⁰⁵Ru,¹⁰⁷Hg, ²⁰³Hg, ^(121m)Te, ^(122m)Te, ^(125m)Te, ¹⁶⁵Tm, ¹⁶⁷Tm, ¹⁶⁸Tm,¹⁹⁷Pt, ¹⁰⁹Pd, ¹⁰⁵Rh, ¹⁴²Pr, ¹⁴³Pr, ¹⁶¹Tb, ¹⁶⁶Ho, ¹⁹⁹Au, ⁵⁷Co, ⁵⁸Co,⁵¹Cr, ⁵⁹Fe, ⁷⁵Se, ²⁰¹Tl, ²²⁵Ac, ⁷⁶Br, ¹⁶⁹Yb, and the like.

Radionuclides and other metals may be delivered, for example, usingchelating groups attached to an antibody or immunoconjugate. Macrocyclicchelates such as NOTA, DOTA, and TETA are of use with a variety ofmetals and radiometals, most particularly with radionuclides of gallium,yttrium and copper, respectively. Such metal-chelate complexes can bemade very stable by tailoring the ring size to the metal of interest.Other ring-type chelates, such as macrocyclic polyethers for complexing²²³Ra, may be used.

Therapeutic agents of use in combination with the antibodies orimmunoconjugates described herein also include, for example,chemotherapeutic drugs such as vinca alkaloids, anthracyclines,epidophyllotoxins, taxanes, antimetabolites, tyrosine kinase inhibitors,Bruton tyrosine kinase inhibitors, microtubule inhibitors, PARPinhibitors, PI3K inhibitors, alkylating agents, antibiotics, Cox-2inhibitors, antimitotics, antiangiogenic and proapoptotic agents,particularly doxorubicin, methotrexate, taxol, other camptothecins, andothers from these and other classes of anticancer agents, and the like.Other cancer chemotherapeutic drugs include nitrogen mustards, alkylsulfonates, nitrosoureas, triazenes, folic acid analogs, pyrimidineanalogs, purine analogs, platinum coordination complexes, hormones, andthe like. Suitable chemotherapeutic agents are described in REMINGTON'SPHARMACEUTICAL SCIENCES, 19th Ed. (Mack Publishing Co. 1995), and inGOODMAN AND GILMAN'S THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, 7th Ed.(MacMillan Publishing Co. 1985), as well as revised editions of thesepublications. Other suitable chemotherapeutic agents, such asexperimental drugs, are known to those of skill in the art.

Exemplary drugs of use include, but are not limited to, 5-fluorouracil,afatinib, aplidin, azaribine, anastrozole, anthracyclines, axitinib,AVL-101, AVL-291, bendamustine, bleomycin, bortezomib, bosutinib,bryostatin-1, busulfan, calicheamycin, camptothecin, carboplatin,10-hydroxycamptothecin, carmustine, celebrex, chlorambucil, cisplatin(CDDP), Cox-2 inhibitors, irinotecan (CPT-11), SN-38, carboplatin,cladribine, camptothecans, crizotinib, cyclophosphamide, cytarabine,dacarbazine, dasatinib, dinaciclib, docetaxel, dactinomycin,daunorubicin, doxorubicin, 2-pyrrolinodoxorubicine (2PDOX), pro-2PDOX,cyano-morpholino doxorubicin, doxorubicin glucuronide, epirubicinglucuronide, erlotinib, estramustine, epidophyllotoxin, erlotinib,entinostat, estrogen receptor binding agents, etoposide (VP16),etoposide glucuronide, etoposide phosphate, exemestane, fingolimod,floxuridine (FUdR), 3′,5′-O-dioleoyl-FudR (FUdR-dO), fludarabine,flutamide, farnesyl-protein transferase inhibitors, flavopiridol,fostamatinib, ganetespib, GDC-0834, GS-1101, gefitinib, gemcitabine,hydroxyurea, ibrutinib, idarubicin, idelalisib, ifosfamide, imatinib,L-asparaginase, lapatinib, lenolidamide, leucovorin, LFM-A13, lomustine,mechlorethamine, melphalan, mercaptopurine, 6-mercaptopurine,methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, navelbine,neratinib, nilotinib, nitrosurea, olaparib, plicomycin, procarbazine,paclitaxel, PCI-32765, pentostatin, PSI-341, raloxifene, semustine,sorafenib, streptozocin, SU11248, sunitinib, tamoxifen, temazolomide (anaqueous form of DTIC), transplatinum, thalidomide, thioguanine,thiotepa, teniposide, topotecan, uracil mustard, vatalanib, vinorelbine,vinblastine, vincristine, vinca alkaloids and ZD1839. Such agents may bepart of the conjugates described herein or may alternatively beadministered in combination with the described conjugates, either priorto, simultaneously with or after the conjugate. Alternatively, one ormore therapeutic naked antibodies as are known in the art may be used incombination with the described conjugates. Exemplary therapeutic nakedantibodies are described above.

In preferred embodiments, a therapeutic agent to be used in combinationwith a DNA-breaking antibody conjugate (e.g., an SN-38-ADC) is amicrotubule inhibitor, such as a vinca alkaloid, a taxanes, amaytansinoid or an auristatin. Exemplary known microtubule inhibitorsinclude paclitaxel, vincristine, vinblastine, mertansine, epothilone,docetaxel, discodermolide, combrestatin, podophyllotoxin, CI-980,phenylahistins, steganacins, curacins, 2-methoxy estradiol, E7010,methoxy benzenesuflonamides, vinorelbine, vinflunine, vindesine,dolastatins, spongistatin, rhizoxin, tasidotin, halichondrins,hemiasterlins, cryptophycin 52, MMAE and eribulin mesylate.

In an alternative preferred embodiment, a therapeutic agent to be usedin combination with a DNA-breaking ADC, such as an SN-38-antibodyconjugate, is a PARP inhibitor, such as olaparib, talazoparib (BMN-673),rucaparib, veliparib, CEP 9722, MK 4827, BGB-290, ABT-888, AG014699,BSI-201, CEP-8983 or 3-aminobenzamide.

In another alternative, a therapeutic agent used in combination with anantibody or immunoconjugate is a Bruton kinase inhibitor, such as suchas ibrutinib (PCI-32765), PCI-45292, CC-292 (AVL-292), ONO-4059,GDC-0834, LFM-A13 or RN486.

In yet another alternative, a therapeutic agent used in combination withan antibody or immunoconjugate is a PI3K inhibitor, such as idelalisib,Wortmannin, demethoxyviridin, perifosine, PX-866, IPI-145 (duvelisib),BAY 80-6946, BEZ235, RP6530, TGR1202, SF1126, INK1117, GDC-0941, BKM120,XL147, XL765, Palomid 529, GSK1059615, ZSTK474, PWT33597, IC_(87114,)TG100-115, CAL263, PI-103, GNE477, CUDC-907, AEZS-136 or LY294002.

Therapeutic agents that may be used in concert with the antibodies orimmunoconjugates also may comprise toxins conjugated to targetingmoieties. Toxins that may be used in this regard include ricin, abrin,ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A, pokeweedantiviral protein, gelonin, diphtheria toxin, Pseudomonas exotoxin, andPseudomonas endotoxin. (See, e.g., Pastan. et al., Cell (1986), 47:641,and Sharkey and Goldenberg, CA Cancer J Clin. 2006 July-August;56(4):226-43.) Additional toxins suitable for use herein are known tothose of skill in the art and are disclosed in U.S. Pat. No. 6,077,499.

Yet another class of therapeutic agent may comprise one or moreimmunomodulators. Immunomodulators of use may be selected from acytokine, a stem cell growth factor, a lymphotoxin, an hematopoieticfactor, a colony stimulating factor (CSF), an interferon (IFN),erythropoietin, thrombopoietin and a combination thereof. Specificallyuseful are lymphotoxins such as tumor necrosis factor (TNF),hematopoietic factors, such as interleukin (IL), colony stimulatingfactor, such as granulocyte-colony stimulating factor (G-CSF) orgranulocyte macrophage-colony stimulating factor (GM-CSF), interferon,such as interferons-α, -β, -γ or -λ, and stem cell growth factor, suchas that designated “S1 factor”. Included among the cytokines are growthhormones such as human growth hormone, N-methionyl human growth hormone,and bovine growth hormone; parathyroid hormone; thyroxine; insulin;proinsulin; relaxin; prorelaxin; glycoprotein hormones such as folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH), andluteinizing hormone (LH); hepatic growth factor; prostaglandin,fibroblast growth factor; prolactin; placental lactogen, OB protein;tumor necrosis factor-β and -ß; mullerian-inhibiting substance; mousegonadotropin-associated peptide; inhibin; activin; vascular endothelialgrowth factor; integrin; thrombopoietin (TPO); nerve growth factors suchas NGF-ß; platelet-growth factor; transforming growth factors (TGFs)such as TGF-α and TGF-ß; insulin-like growth factor-I and -II;erythropoietin (EPO); osteoinductive factors; interferons such asinterferon-α, -β, and -γ; colony stimulating factors (CSFs) such asmacrophage-CSF (M-CSF); interleukins (ILs) such as IL-1, IL-1α, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-13,IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-25, LIF, kit-ligand orFLT-3, angiostatin, thrombospondin, endostatin, tumor necrosis factorand lymphotoxin (LT). As used herein, the term cytokine includesproteins from natural sources or from recombinant cell culture andbiologically active equivalents of the native sequence cytokines.

Chemokines of use include RANTES, MCAF, MIP1-alpha, MIP1-Beta and IP-10.

The person of ordinary skill will realize that the subject antibodies orimmunoconjugates may be used alone or in combination with one or moreother therapeutic agents, such as a second antibody, second antibodyfragment, second immunoconjugate, radionuclide, toxin, drug,chemotherapeutic agent, radiation therapy, chemokine, cytokine,immunomodulator, enzyme, hormone, oligonucleotide, RNAi or siRNA.Preferably, the therapeutic agent is a PARP inhibitor, a microtubuleinhibitor, a Bruton kinase inhibitor or a PI3K inhibitor. Exemplaryknown PARP inhibitors include olaparib, talazoparib (BMN-673),rucaparib, veliparib, CEP 9722, MK 4827, BGB-290, ABT-888, AG014699,BSI-201, CEP-8983 or 3-aminobenzamide. Exemplary known microtubuleinhibitors include, but are not limited to, vinca alkaloid, taxanes,maytansinoids, auristatins, paclitaxel, vincristine, vinblastine,mertansine, epothilone, docetaxel, discodermolide, combrestatin,podophyllotoxin, CI-980, phenylahistins, steganacins, curacins,2-methoxy estradiol, E7010, methoxy benzenesuflonamides, vinorelbine,vinflunine, vindesine, dolastatins, spongistatin, rhizoxin, tasidotin,halichondrins, hemiasterlins, cryptophycin 52, MMAE and eribulinmesylate. Exemplary Bruton kinase inhibitors include ibrutinib(PCI-32765), PCI-45292, CC-292 (AVL-292), ONO-4059, GDC-0834, LFM-A13 orRN486. Exemplary PI3K inhibitors include idelalisib, Wortmannin,demethoxyviridin, perifosine, PX-866, IPI-145 (duvelisib), BAY 80-6946,BEZ235, RP6530, TGR1202, SF1126, INK1117, GDC-0941, BKM120, XL147,XL765, Palomid 529, GSK1059615, ZSTK474, PWT33597, IC_(87114,)TG100-115, CAL263, PI-103, GNE477, CUDC-907, AEZS-136 or LY294002. Suchadditional therapeutic agents may be administered separately, incombination with, or attached to the subject antibodies orimmunoconjugates.

Formulation and Administration

Suitable routes of administration of the antibodies, immunoconjugatesand/or drugs include, without limitation, parenteral, subcutaneous,rectal, transmucosal, intestinal administration, intramuscular,intramedullary, intrathecal, direct intraventricular, intravenous,intravitreal, intraperitoneal, intranasal, or intraocular injections.The preferred routes of administration are parenteral. Alternatively,one may administer the compound in a local rather than systemic manner,for example, via injection of the compound directly into a solid tumor.Certain drugs, such as microtubule inhibitors, PARP inhibitors, Brutonkinase inhibitors or PI3K inhibitors may be designed to be administeredorally.

Antibodies or immunoconjugates can be formulated according to knownmethods to prepare pharmaceutically useful compositions, whereby theantibody or immunoconjugate is combined in a mixture with apharmaceutically suitable excipient. Sterile phosphate-buffered salineis one example of a pharmaceutically suitable excipient. Other suitableexcipients are well-known to those in the art. See, for example, Anselet al., PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5thEdition (Lea & Febiger 1990), and Gennaro (ed.), REMINGTON'SPHARMACEUTICAL SCIENCES, 18th Edition (Mack Publishing Company 1990),and revised editions thereof.

In a preferred embodiment, the antibody or immunoconjugate is formulatedin Good's biological buffer (pH 6-7), using a buffer selected from thegroup consisting of N-(2-acetamido)-2-aminoethanesulfonic acid (ACES);N-(2-acetamido)iminodiacetic acid (ADA);N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES);4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES);2-(N-morpholino)ethanesulfonic acid (IVIES);3-(N-morpholino)propanesulfonic acid (MOPS);3-(N-morpholinyl)-2-hydroxypropanesulfonic acid (MOPSO); andpiperazine-N,N′-bis(2-ethanesulfonic acid) [Pipes]. More preferredbuffers are MES or MOPS, preferably in the concentration range of 20 to100 mM, more preferably about 25 mM. Most preferred is 25 mM IVIES, pH6.5. The formulation may further comprise 25 mM trehalose and 0.01% v/vpolysorbate 80 as excipients, with the final buffer concentrationmodified to 22.25 mM as a result of added excipients. The preferredmethod of storage is as a lyophilized formulation of the conjugates,stored in the temperature range of −20° C. to 2° C., with the mostpreferred storage at 2° C. to 8° C.

The antibody or immunoconjugate can be formulated for intravenousadministration via, for example, bolus injection, slow infusion orcontinuous infusion. Preferably, the antibody of the present inventionis infused over a period of less than about 4 hours, and morepreferably, over a period of less than about 3 hours. For example, thefirst 25-50 mg could be infused within 30 minutes, preferably even 15min, and the remainder infused over the next 2-3 hrs. Formulations forinjection can be presented in unit dosage form, e.g., in ampoules or inmulti-dose containers, with an added preservative. The compositions cantake such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and can contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient can be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

Additional pharmaceutical methods may be employed to control theduration of action of the therapeutic conjugate. Control releasepreparations can be prepared through the use of polymers to complex oradsorb the immunoconjugate. For example, biocompatible polymers includematrices of poly(ethylene-co-vinyl acetate) and matrices of apolyanhydride copolymer of a stearic acid dimer and sebacic acid.Sherwood et al., Bio/Technology 10: 1446 (1992). The rate of release ofan antibody or immunoconjugate from such a matrix depends upon themolecular weight, the amount of antibody or immunoconjugate within thematrix, and the size of dispersed particles. Saltzman et al., Biophys.J. 55: 163 (1989); Sherwood et al., supra. Other solid dosage forms aredescribed in Ansel et al., PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERYSYSTEMS, 5th Edition (Lea & Febiger 1990), and Gennaro (ed.),REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition (Mack PublishingCompany 1990), and revised editions thereof.

Generally, the dosage of an administered antibody or immunoconjugate forhumans will vary depending upon such factors as the patient's age,weight, height, sex, general medical condition and previous medicalhistory. It may be desirable to provide the recipient with a dosage ofimmunoconjugate that is in the range of from about 1 mg/kg to 24 mg/kgas a single intravenous infusion, although a lower or higher dosage alsomay be administered as circumstances dictate. A dosage of 1-20 mg/kg fora 70 kg patient, for example, is 70-1,400 mg, or 41-824 mg/m² for a1.7-m patient. The dosage may be repeated as needed, for example, onceper week for 4-10 weeks, once per week for 8 weeks, or once per week for4 weeks. It may also be given less frequently, such as every other weekfor several months, or monthly or quarterly for many months, as neededin a maintenance therapy. Preferred dosages may include, but are notlimited to, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, 11 mg/kg, 12 mg/kg, 13 mg/kg, 14mg/kg, 15 mg/kg, 16 mg/kg, 17 mg/kg, and 18 mg/kg. The dosage ispreferably administered multiple times, once or twice a week, or asinfrequently as once every 3 or 4 weeks. A minimum dosage schedule of 4weeks, more preferably 8 weeks, more preferably 16 weeks or longer maybe used. The schedule of administration may comprise administration onceor twice a week, on a cycle selected from the group consisting of: (i)weekly; (ii) every other week; (iii) one week of therapy followed bytwo, three or four weeks off; (iv) two weeks of therapy followed by one,two, three or four weeks off; (v) three weeks of therapy followed byone, two, three, four or five week off; (vi) four weeks of therapyfollowed by one, two, three, four or five week off; (vii) five weeks oftherapy followed by one, two, three, four or five week off; (viii)monthly and (ix) every 3 weeks. The cycle may be repeated 2, 4, 6, 8,10, 12, 16 or 20 times or more.

Alternatively, an antibody or immunoconjugate may be administered as onedosage every 2 or 3 weeks, repeated for a total of at least 3 dosages.Or, twice per week for 4-6 weeks. If the dosage is lowered toapproximately 200-300 mg/m² (340 mg per dosage for a 1.7-m patient, or4.9 mg/kg for a 70 kg patient), it may be administered once or eventwice weekly for 4 to 10 weeks. Alternatively, the dosage schedule maybe decreased, namely every 2 or 3 weeks for 2-3 months. It has beendetermined, however, that even higher doses, such as 12 mg/kg onceweekly or once every 2-3 weeks can be administered by slow i.v.infusion, for repeated dosing cycles. The dosing schedule can optionallybe repeated at other intervals and dosage may be given through variousparenteral routes, with appropriate adjustment of the dose and schedule.

In preferred embodiments, the antibodies or immunoconjugates are of usefor therapy of cancer. Examples of cancers include, but are not limitedto, carcinoma, lymphoma, glioblastoma, melanoma, sarcoma, and leukemia,myeloma, or lymphoid malignancies. More particular examples of suchcancers are noted below and include: squamous cell cancer (e.g.,epithelial squamous cell cancer), Ewing sarcoma, Wilms tumor,astrocytomas, glioblastomas, lung cancer including small-cell lungcancer, non-small cell lung cancer, adenocarcinoma of the lung andsquamous carcinoma of the lung, cancer of the peritoneum, gastric orstomach cancer including gastrointestinal cancer, pancreatic cancer,glioblastoma multiforme, cervical cancer, ovarian cancer, liver cancer,bladder cancer, hepatoma, hepatocellular carcinoma, neuroendocrinetumors, medullary thyroid cancer, differentiated thyroid carcinoma,breast cancer, ovarian cancer, colon cancer, rectal cancer, endometrialcancer or uterine carcinoma, salivary gland carcinoma, kidney or renalcancer, prostate cancer, vulvar cancer, anal carcinoma, penilecarcinoma, as well as head-and-neck cancer. The term “cancer” includesprimary malignant cells or tumors (e.g., those whose cells have notmigrated to sites in the subject's body other than the site of theoriginal malignancy or tumor) and secondary malignant cells or tumors(e.g., those arising from metastasis, the migration of malignant cellsor tumor cells to secondary sites that are different from the site ofthe original tumor).

Other examples of cancers or malignancies include, but are not limitedto: Acute Childhood Lymphoblastic Leukemia, Acute LymphoblasticLeukemia, Acute Lymphocytic Leukemia, Acute Myeloid Leukemia,Adrenocortical Carcinoma, Adult (Primary) Hepatocellular Cancer, Adult(Primary) Liver Cancer, Adult Acute Lymphocytic Leukemia, Adult AcuteMyeloid Leukemia, Adult Hodgkin's Lymphoma, Adult Lymphocytic Leukemia,Adult Non-Hodgkin's Lymphoma, Adult Primary Liver Cancer, Adult SoftTissue Sarcoma, AIDS-Related Lymphoma, AIDS-Related Malignancies, AnalCancer, Astrocytoma, Bile Duct Cancer, Bladder Cancer, Bone Cancer,Brain Stem Glioma, Brain Tumors, Breast Cancer, Cancer of the RenalPelvis and Ureter, Central Nervous System (Primary) Lymphoma, CentralNervous System Lymphoma, Cerebellar Astrocytoma, Cerebral Astrocytoma,Cervical Cancer, Childhood (Primary) Hepatocellular Cancer, Childhood(Primary) Liver Cancer, Childhood Acute Lymphoblastic Leukemia,Childhood Acute Myeloid Leukemia, Childhood Brain Stem Glioma, ChildhoodCerebellar Astrocytoma, Childhood Cerebral Astrocytoma, ChildhoodExtracranial Germ Cell Tumors, Childhood Hodgkin's Disease, ChildhoodHodgkin's Lymphoma, Childhood Hypothalamic and Visual Pathway Glioma,Childhood Lymphoblastic Leukemia, Childhood Medulloblastoma, ChildhoodNon-Hodgkin's Lymphoma, Childhood Pineal and Supratentorial PrimitiveNeuroectodermal Tumors, Childhood Primary Liver Cancer, ChildhoodRhabdomyosarcoma, Childhood Soft Tissue Sarcoma, Childhood VisualPathway and Hypothalamic Glioma, Chronic Lymphocytic Leukemia, ChronicMyelogenous Leukemia, Colon Cancer, Cutaneous T-Cell Lymphoma, EndocrinePancreas Islet Cell Carcinoma, Endometrial Cancer, Ependymoma,Epithelial Cancer, Esophageal Cancer, Ewing's Sarcoma and RelatedTumors, Exocrine Pancreatic Cancer, Extracranial Germ Cell Tumor,Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Eye Cancer,Female Breast Cancer, Gaucher's Disease, Gallbladder Cancer, GastricCancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Tumors, GermCell Tumors, Gestational Trophoblastic Tumor, Hairy Cell Leukemia, Headand Neck Cancer, Hepatocellular Cancer, Hodgkin's Lymphoma,Hypergammaglobulinemia, Hypopharyngeal Cancer, Intestinal Cancers,Intraocular Melanoma, Islet Cell Carcinoma, Islet Cell PancreaticCancer, Kaposi's Sarcoma, Kidney Cancer, Laryngeal Cancer, Lip and OralCavity Cancer, Liver Cancer, Lung Cancer, Lymphoproliferative Disorders,Macroglobulinemia, Male Breast Cancer, Malignant Mesothelioma, MalignantThymoma, Medulloblastoma, Melanoma, Mesothelioma, Metastatic OccultPrimary Squamous Neck Cancer, Metastatic Primary Squamous Neck Cancer,Metastatic Squamous Neck Cancer, Multiple Myeloma, MultipleMyeloma/Plasma Cell Neoplasm, Myelodysplastic Syndrome, MyelogenousLeukemia, Myeloid Leukemia, Myeloproliferative Disorders, Nasal Cavityand Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma,Non-Hodgkin's Lymphoma, Nonmelanoma Skin Cancer, Non-Small Cell LungCancer, Occult Primary Metastatic Squamous Neck Cancer, OropharyngealCancer, Osteo-/Malignant Fibrous Sarcoma, Osteosarcoma/Malignant FibrousHistiocytoma, Osteosarcoma/Malignant Fibrous Histiocytoma of Bone,Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor, Ovarian LowMalignant Potential Tumor, Pancreatic Cancer, Paraproteinemias,Polycythemia vera, Parathyroid Cancer, Penile Cancer, Pheochromocytoma,Pituitary Tumor, Primary Central Nervous System Lymphoma, Primary LiverCancer, Prostate Cancer, Rectal Cancer, Renal Cell Cancer, Renal Pelvisand Ureter Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary GlandCancer, Sarcoidosis Sarcomas, Sezary Syndrome, Skin Cancer, Small CellLung Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous NeckCancer, Stomach Cancer, Supratentorial Primitive Neuroectodermal andPineal Tumors, T-Cell Lymphoma, Testicular Cancer, Thymoma, ThyroidCancer, Transitional Cell Cancer of the Renal Pelvis and Ureter,Transitional Renal Pelvis and Ureter Cancer, Trophoblastic Tumors,Ureter and Renal Pelvis Cell Cancer, Urethral Cancer, Uterine Cancer,Uterine Sarcoma, Vaginal Cancer, Visual Pathway and Hypothalamic Glioma,Vulvar Cancer, Waldenstrom's macroglobulinemia, Wilms' tumor, and anyother hyperproliferative disease, besides neoplasia, located in an organsystem listed above.

The methods and compositions described and claimed herein may be used totreat malignant or premalignant conditions and to prevent progression toa neoplastic or malignant state, including but not limited to thosedisorders described above. Such uses are indicated in conditions knownor suspected of preceding progression to neoplasia or cancer, inparticular, where non-neoplastic cell growth consisting of hyperplasia,metaplasia, or most particularly, dysplasia has occurred (for review ofsuch abnormal growth conditions, see Robbins and Angell, BasicPathology, 2d Ed., W. B. Saunders Co., Philadelphia, pp. 68-79 (1976)).

Dysplasia is frequently a forerunner of cancer, and is found mainly inthe epithelia. It is the most disorderly form of non-neoplastic cellgrowth, involving a loss in individual cell uniformity and in thearchitectural orientation of cells. Dysplasia characteristically occurswhere there exists chronic irritation or inflammation. Dysplasticdisorders which can be treated include, but are not limited to,anhidrotic ectodermal dysplasia, anterofacial dysplasia, asphyxiatingthoracic dysplasia, atriodigital dysplasia, bronchopulmonary dysplasia,cerebral dysplasia, cervical dysplasia, chondroectodermal dysplasia,cleidocranial dysplasia, congenital ectodermal dysplasia,craniodiaphysial dysplasia, craniocarpotarsal dysplasia,craniometaphysial dysplasia, dentin dysplasia, diaphysial dysplasia,ectodermal dysplasia, enamel dysplasia, encephalo-ophthalmic dysplasia,dysplasia epiphysialis hemimelia, dysplasia epiphysialis multiplex,dysplasia epiphysialis punctata, epithelial dysplasia,faciodigitogenital dysplasia, familial fibrous dysplasia of jaws,familial white folded dysplasia, fibromuscular dysplasia, fibrousdysplasia of bone, florid osseous dysplasia, hereditary renal-retinaldysplasia, hidrotic ectodermal dysplasia, hypohidrotic ectodermaldysplasia, lymphopenic thymic dysplasia, mammary dysplasia,mandibulofacial dysplasia, metaphysial dysplasia, Mondini dysplasia,monostotic fibrous dysplasia, mucoepithelial dysplasia, multipleepiphysial dysplasia, oculoauriculovertebral dysplasia,oculodentodigital dysplasia, oculovertebral dysplasia, odontogenicdysplasia, opthalmomandibulomelic dysplasia, periapical cementaldysplasia, polyostotic fibrous dysplasia, pseudoachondroplasticspondyloepiphysial dysplasia, retinal dysplasia, septo-optic dysplasia,spondyloepiphysial dysplasia, and ventriculoradial dysplasia.

Additional pre-neoplastic disorders which can be treated include, butare not limited to, benign dysproliferative disorders (e.g., benigntumors, fibrocystic conditions, tissue hypertrophy, intestinal polyps oradenomas, and esophageal dysplasia), leukoplakia, keratoses, Bowen'sdisease, Farmer's Skin, solar cheilitis, and solar keratosis.

In preferred embodiments, the method of the invention is used to inhibitgrowth, progression, and/or metastasis of cancers, in particular thoselisted above.

Additional hyperproliferative diseases, disorders, and/or conditionsinclude, but are not limited to, progression, and/or metastases ofmalignancies and related disorders such as leukemia (including acuteleukemias; e.g., acute lymphocytic leukemia, acute myelocytic leukemia[including myeloblastic, promyelocytic, myelomonocytic, monocytic, anderythroleukemia]) and chronic leukemias (e.g., chronic myelocytic[granulocytic] leukemia and chronic lymphocytic leukemia), polycythemiavera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease),multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease,and solid tumors including, but not limited to, sarcomas and carcinomassuch as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor,cervical cancer, testicular tumor, lung carcinoma, small cell lungcarcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,emangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,melanoma, neuroblastoma, and retinoblastoma.

Autoimmune diseases that may be treated with antibodies orimmunoconjugates may include acute and chronic immune thrombocytopenias,dermatomyositis, Sydenham's chorea, myasthenia gravis, systemic lupuserythematosus, lupus nephritis, rheumatic fever, polyglandularsyndromes, bullous pemphigoid, diabetes mellitus, Henoch-Schonleinpurpura, post-streptococcal nephritis, erythema nodosum, Takayasu'sarteritis, ANCA-associated vasculitides, Addison's disease, rheumatoidarthritis, multiple sclerosis, sarcoidosis, ulcerative colitis, erythemamultiforme, IgA nephropathy, polyarteritis nodosa, ankylosingspondylitis, Goodpasture's syndrome, thromboangitis obliterans,Sjögren's syndrome, primary biliary cirrhosis, Hashimoto's thyroiditis,thyrotoxicosis, scleroderma, chronic active hepatitis,polymyositis/dermatomyositis, polychondritis, bullous pemphigoid,pemphigus vulgaris, Wegener's granulomatosis, membranous nephropathy,amyotrophic lateral sclerosis, tabes dorsalis, giant cellarteritis/polymyalgia, pernicious anemia, rapidly progressiveglomerulonephritis, psoriasis or fibrosing alveolitis.

Kits

Various embodiments may concern kits containing components suitable fortreating diseased tissue in a patient. Exemplary kits may contain atleast one antibody or immunoconjugate as described herein. A kit mayalso include a drug selected from microtubule inhibitors, PARPinhibitors, Bruton kinase inhibitors and/or PI3K inhibitors. If thecomposition containing components for administration is not formulatedfor delivery via the alimentary canal, such as by oral delivery, adevice capable of delivering the kit components through some other routemay be included. One type of device, for applications such as parenteraldelivery, is a syringe that is used to inject the composition into thebody of a subject. Inhalation devices may also be used.

The kit components may be packaged together or separated into two ormore containers. In some embodiments, the containers may be vials thatcontain sterile, lyophilized formulations of a composition that aresuitable for reconstitution. A kit may also contain one or more bufferssuitable for reconstitution and/or dilution of other reagents. Othercontainers that may be used include, but are not limited to, a pouch,tray, box, tube, or the like. Kit components may be packaged andmaintained sterilely within the containers. Another component that canbe included is instructions to a person using a kit for its use.

EXAMPLES

Various embodiments of the present invention are illustrated by thefollowing examples, without limiting the scope thereof.

Example 1 Combination Therapy with ADC IMMU-132 and MicrotubuleInhibitors or PARP Inhibitors

In current clinical trials (ClinicalTrials.gov, NCT01631552),triple-negative breast cancer (TNBC) patients treated with IMMU-132,which is composed of the active metabolite of irinotecan, SN-38,conjugated to an anti-Trop-2 antibody, shows manageable toxicity andvery encouraging responses in relapsed/refractory cases.

Synthetic lethality is a concept in which a cell harboring one out oftwo possible gene or protein defects is viable, while a cell containingboth defects is nonviable. BRCA1/2 mutations are linked to deficienciesin DNA repair and are associated with TNBC. Other repair mechanismsinvolve poly(adenosine diphosphoribose) polymerase (PARP), which can beused by cancer cells to overcome loss of BRACA1/2. Treatment of TNBCcells with either IMMU-132 or paclitaxel results in cleavage anddeactivation of PARP, whereas the small molecule olaparib directlyinhibits PARP. Therefore, the rationale of combining IMMU-132 witheither paclitaxel or olaparib to effectively knock-out PARP activity wasinvestigated in TNBC xenografts to ascertain if these combinations willresult in synthetic lethality.

The purpose of this study was to determine whether combining anantibody-drug conjugate that induces DNA strand breaks, such assacituzumab govitecan (also known as IMMU-132, an anti-Trop-2hRS7-CL2A-SN-38), with microtubule inhibitors (e.g., paclitaxel oreribulin mesylate) or poly(adenosine diphosphoribose) polymerase (PARP)inhibitors (e.g., olaparib) in cancer (e.g., nude mice bearing TNBCxenografts) improves anti-tumor effects. The person of ordinary skillwill realize that the unexpected superior effects of the combination ofantibody-SN-38 conjugates with PARP or microtubule inhibitors are notlimited to the specific exemplary antibody, drug, PARP inhibitor ormicrotubule inhibitor, but rather are characteristic of the classes ofantibodies against tumor-associated antigens (TAAs), drugs that induceDNA strand breaks, PARP inhibitors and microtubule inhibitors.

Experimental Procedures

In a non-limiting example, mice bearing human TNBC (triple negativebreast cancer) xenografts (MDA-MB-468 or HCC1806; ˜0.3 cm³) were treatedwith the maximum tolerated dose of paclitaxel (15 mg/kg weekly×5 wks)and IMMU-132 at either 10 mg/kg or 12.5 mg/kg on days 1, 8, 22, and 29.Mice bearing HCC1806 tumors (˜0.28 cm³) were treated for 2 cycles withIMMU-132 (12.5 mg/kg) and 0.5 mg/kg of eribulin mesylate (equivalent tohuman dose of 1.4 mg/m²) weekly for 2 weeks on a 21-day cycle. Studiesexamining PARP inhibition used mice bearing MDA-MB-468 tumors (˜0.32cm³) treated with olaparib (50 mg/kg, qdx5d, ×4 wks; 33% of human doseequaling 800 mg daily) and IMMU-132 (10 mg/kg, twice weekly×4 wks).Olaparib was administered as i.p. injections daily for 5 days in a rowwith two day's rest before repeating (qdx5). This was done for fourweeks. IMMU-132 was administered i.p. twice weekly for four weeks.Control animals received the non-tumor targeting anti-CD20 ADChA20-CL2A-SN-38, either alone or in combination with olaparib. Theprimary endpoint was the median survival time (MST), defined as the timefor tumors to progress to 1.0 cm³.

In alternative embodiments, assay for synergistic effects may bedetermined by in vitro assay. A clonogenic assay may be used todetermine survival fraction of cells (Ibrahim et al., 2012, CancerDiscovery 2:1036-47). Briefly, 350-800 cells are plated in 6-well flatbottom cell culture plates in duplicates. Twenty-four hours afterplating, cells are washed and fresh medium is added in the presence orabsence of increasing doses of ADC and/or PARP or microtubule inhibitor(e.g., olaparib) alone and in combination. Media containing the drugand/or is refreshed on day 4. Colonies are fixed and stained after 7days of treatment with 1.5 ml of 6.0% glutaraldehyde and 0.5% crystalviolet and colonies are counted by standard procedures. The survivingfraction (SF) of cells is calculated as follows:

${SF} = \frac{{Number}\mspace{14mu}{of}\mspace{14mu}{colonies}\mspace{14mu}{formed}\mspace{14mu}{after}\mspace{14mu}{treatment}}{{Number}\mspace{14mu}{of}\mspace{14mu}{cells}\mspace{14mu}{seeded} \times {Plating}\mspace{14mu}{Efficiency}}$${{where}\mspace{14mu}{Plating}\mspace{14mu}{Efficiency}} = \frac{{Number}\mspace{14mu}{of}\mspace{14mu}{colonies}\mspace{14mu}{formed}\mspace{14mu}{in}\mspace{14mu}{control}}{{Number}\mspace{14mu}{of}\mspace{14mu}{cells}\mspace{14mu}{seeded}}$

The interaction between ADC and PARP or microtubule inhibitor isassessed using the multiple drug effects analysis method of Chou andTalalay (1984, Adv Enzyme Regul 22:27-55). This method quantitativelydescribes the interaction between two or more drugs, with values lessthan 1 indicating synergistic interactions, values greater than 1indicating antagonistic interactions, and values equal to 1 indicatingadditive interactions.

Results

Mice with MDA-MB-468 tumors given the combination of IMMU-132 andpaclitaxel exhibited superior anti-tumor effects (FIG. 1A-1C),with >11-fold tumor shrinkage, in comparison to 1.4-fold shrinkage inthe IMMU-132 group alone (P=0.0003; area under the curve, AUC) or11.4-fold increase in tumor size in mice treated with paclitaxel alone(P<0.0001; AUC).

In MDA-MB-468, the combination of 200 μg IMMU-132 plus paclitaxel hassuperior anti-tumor effects in terms of area under the curve (AUC) whencompared to all the other groups (Table 7, P<0.0013). Lowering theamount of IMMU-132 administered with paclitaxel to 100 μg likewiseresults in significant anti-tumor effects as compared to mice treatedwith paclitaxel alone, IMMU-132 alone (100 μg), or untreated animals(Table 8, P<0.0328). No further comparisons between growth curves can bemade with paclitaxel or untreated control groups since each began tolose mice due to disease progression (i.e., TV>1.0 cm³) as of therapyday 49.

TABLE 7 Area under the curve comparisons between IMMU-132 (200 μg) plusPaclitaxel treated MDA-MB-468 tumor-bearing mice and all other treatmentgroups. Tumor Volumes (cm³) on Time of that day P-Value TreatmentsComparison (mean ± s.d.) (AUC) IMMU- IMMU-132 Up to therapy 0.162 ±0.144 vs. 0.0003 132 (200 μg) Day 98 0.621 ± 0.324    (200 μg) IMMU-132Up to therapy 0.050 ± 0.062 vs. 0.0002 plus (100 μg) Day 70 0.634 ±0.335    Paclitaxel Paclitaxel Up to therapy 0.025 ± 0.041 vs. <0.0001versus Day 49 0.705 ± 0.206    IMMU-132 Up to therapy 0.202 ± 0.191 vs.0.0013 (100 μg) + Day 112 0.496 ± 0.286    Paclitaxel Untreated Up totherapy 0.025 ± 0.041 vs. <0.0001 Day 49 0.663 ± 0.349   

TABLE 8 Area under the curve comparisons between IMMU-132 (100 μg) plusPaclitaxel treated MDA-MB-468 tumor-bearing mice and all other treatmentgroups. Tumor Volumes (cm³) on Time of that day P-Value TreatmentsComparison (mean ± s.d.) (AUC) IMMU- IMMU-132 Up to therapy 0.663 ±0.349 vs. 0.9539 132 (200 ug) Day 98 0.621 ± 0.324    (100 μg) IMMU-132Up to therapy 0.311 ± 0.196 vs. 0.0328 plus (100 μg) Day 70 0.634 ±0.335    Paclitaxel Paclitaxel Up to therapy 0.211 ± 0.155 vs. <0.0001versus Day 49 0.705 ± 0.206    Untreated Up to therapy 0.211 ± 0.155 vs.0.0001 Day 49 0.663 ± 0.349   

In the rapidly-progressing HCC1806 xenografts (FIG. 2A-2B), thecombination of IMMU-132 plus paclitaxel proved to have a superioranti-tumor effect when compared to IMMU-132 monotherapy (P=0.0195,AUC_(17days)). This is a very aggressive tumor with a median survivaltime (MST) of only 10 days post-therapy initiation for the untreatedcontrol animals (18 days post-tumor cell inoculation). In terms ofsurvival, the combination, which reached its MST of 38 days, provided asignificant survival benefit when compared to all other therapies(P<0.017; log-rank). It should be noted that this was achieved at a lowdose of only 0.25 mg which would be the human equivalent dose of only 1mg/kg.

Mice treated with the combination of IMMU-132 plus eribulin mesylate(FIG. 3A-3E) exhibited a significantly greater anti-tumor response thanall other monotherapy groups (P<0.0432; paired t-test). This resulted ina significant survival benefit for the combination (MST=23 days) whencompared to eribulin or IMMU-132 monotherapy (MST=18 and 14 days,respectively; P<0.0044; log-rank).

Likewise, combining IMMU-132 therapy with olaparib was superior tosingle agent therapy in mice bearing MDA-MB-468 tumors (P<0.0032; AUC)(FIG. 4). Results are summarized in Table 9. All the IMMU-132combination treatments were well-tolerated.

TABLE 9 Area under the curve comparisons between IMMU-132 plus Olaparibtreated MDA-MB-468 tumor-bearing mice and all other treatment groups.Tumor Volumes (cm³) Time of on that day P-Value Treatments Comparison(mean ± s.d.) (AUC) IMMU- IMMU-132 Up to therapy 0.030 ± 0.038 vs.0.0023 132 plus Alone Day 49 0.088 ± 0.069    Olaparib hA20-SN-38 Up totherapy 0.030 ± 0.038 vs. <0.0001 versus plus Olaparib Day 49 0.652 ±0.306    hA20-SN-38 Up to therapy 0.045 ± 0.045 vs. 0.0002 Alone Day 420.654 ± 0.285    Olaparib Up to therapy 0.083 ± 0.050 vs. <0.0001 AloneDay 28 0.649 ± 0.267    Saline Up to therapy 0.083 ± 0.050 vs. <0.0001Day 28 0.697 ± 0.352   

Drug-antibody ratio (DAR) determination. Five clinical lots of IMMU-132were evaluated by hydrophobic interaction HPLC (HIC-HPLC), whichresolved three peaks representing species with DARs of 6, 7 and 8, withthe greatest fraction comprising a DAR=8 (not shown). IMMU-132 wasproduced consistently by this manufacturing process, with an overall DAR(DARAvE) of 7.60±0.03 among the five clinical lots. HIC-HPLC resultswere confirmed by liquid chromatography-mass spectrometry (LC-MS). Theanalysis showed that >99% of the 8 available sulfhydryl groups werecoupled with the CL2A linker, either with or without SN-38 (not shown).There were no unsubstituted (or N-ethylmaleimide capped) heavy or lightchains detected. Thus, the difference in DAR among the species resultsfrom SN-38 liberation from the linker during manufacturing and not froma lower initial substitution ratio. Once prepared and lyophilized,IMMU-132 has been stable for several years.

Effect of DAR on pharmacokinetics and anti-tumor efficacy in mice. Micebearing Trop-2⁺ human gastric carcinoma xenografts (NCI-N87) were given2 treatments 7 days apart, each with equal protein (0.5 mg) doses ofIMMU-132 having DARs of 6.89, 3.28, or 1.64. Animals treated with theADCs having a DAR of 6.89 had a significantly improved median survivaltime (MST) compared to mice given ADCs with either 3.38 or 1.64 DARs(MST=39 days vs. 25 and 21 days, respectively; P<0.0014) (not shown).There was no difference between groups treated with the 3.28 or 1.64 DARconjugates and the saline control group.

To further elucidate the importance of a higher DAR, mice bearingNCI-N87 gastric tumors were administered 0.5 mg IMMU-132 with a DAR of6.89 twice weekly for two weeks (not shown). Another group receivedtwice the protein (1 mg) dose of an IMMU-132 conjugate with a DAR of3.28. Although both groups received the same total amount of SN-38 (36μg) with each dosing scheme, those treated with the 6.89 DAR conjugateinhibited tumor growth significantly more than tumor-bearing animalstreated with the 3.28 DAR conjugate (P=0.0227; AUC) (not shown).Additionally, treatment with the lower DAR was not significantlydifferent than the untreated controls. Collectively, these studiesindicate that a lower DAR reduces efficacy.

An examination of the pharmacokinetic behavior of conjugates prepared atthese different ratios was performed in non-tumor-bearing mice given 0.2mg of each conjugate, unconjugated hRS7 IgG, or hRS7 IgG that wasreduced and then capped with N-ethylmaleimide. Serum was taken at 5intervals from 0.5 to 168 h and assayed by ELISA for hRS7 IgG. There wasno significant difference in the clearance of these conjugates comparedto the unconjugated IgG (not shown). Thus, the substitution level didnot affect the pharmacokinetics of the conjugates, and equallyimportant, the reduction of the interchain disulfide bonds did notappear to destabilize the antibody.

Mechanism of action of IMMU-132 in TNBC. The apoptotic pathway utilizedby IMMU-132 was examined in the TNBC cell line, MDA-MB-468, and in theHER2⁺ SK-BR-3 cell line, in order to confirm that the ADC functions onthe basis of its incorporated SN-38. Cells were exposed to 1 μM SN-38,the SN-38-equivalent of IMMU-132, or protein equivalent of hRS7. Cellswere harvested and Western blots were performed. SN-38 alone andIMMU-132 mediated >2-fold up-regulation of p21^(WAF1/Cip1) within 24 hin MDA-MB-468, and by 48 h, the amount of p21^(WAF1/Cip1) in these cellsbegan to decrease (31% and 43% with SN-38 or IMMU-132, respectively)(not shown). Interestingly, in the HER2⁺ SK-BR-3 tumor line, neitherSN-38 nor IMMU-132 mediated the up-regulation of p21^(WAF1/Cip1) aboveconstitutive levels in the first 24 h, but as seen in MDA-MB-468 cellsafter 48-h exposure to SN-38 or IMMU-132, the amount of p21^(WAF1/Cip1)decreased >57% (not shown). Both SN-38 and IMMU-132 resulted in cleavageof pro-caspase-3 into its active fragments within 24 h, but with thegreater degree of active fragments observed after exposure for 48 h. Ofnote, in both cell lines, IMMU-132 mediated a greater degree ofpro-caspase-3 cleavage, with the highest level observed after 48 h whencompared to cells exposed to SN-38 (not shown). Finally, SN-38 andIMMU-132 both mediated poly ADP ribose polymerase (PARP) cleavage,starting at 24 h, with near complete cleavage after 48 h (not shown).Taken together, these results confirm that IMMU-132 has a mechanism ofaction similar to that of free SN-38 when administered in vitro.

Delivery of SN-38 by IMMU-132 vs. irinotecan in a human tumor xenograftmodel. Constitutive products derived from irinotecan or IMMU-132 weredetermined in the serum and tumors of mice implanted s.c. with a humanpancreatic cancer xenograft (Capan-1) administered irinotecan (773 μg;SN-38 equivalents=448 μg) and IMMU-132 (1.0 mg; SN-38 equivalents=16μg). Following administration, at 5 intervals 3 animals from each groupwere euthanized with serum extracted for the products of interest.

Irinotecan cleared very rapidly from serum, with conversion to SN-38 andSN-38G seen within 5 min (not shown). None of the products was detectedat 24 h. The AUCs over a 6-h period were 21.0, 2.5, and 2.8 μg/mL·h foririnotecan, SN-38, and SN-38G, respectively (SN-38 conversion inmice=[2.5+2.8)/21=25.2%]). Animals given IMMU-132 had much lowerconcentrations of free SN-38 in the serum, but it was detected through48 h (not shown). Free SN-38G was detected only at 1 and 6 h, and was 3-to 7-times lower than free SN-38 (not shown).

In the Capan-1 tumors excised from irinotecan-treated animals,irinotecan levels were high over 6 h, but undetectable a 24 h(AUC_(5min-6 h)=48.4 μg/g·h). SN-38 was much lower and detected onlythrough 2 h (i.e., AUC_(5min-2 h)=0.4 μg/g·h), with SN-38G values almost3-fold higher (AUC=1.1 μg/g·h) (not shown). Tumors taken from animalsgiven IMMU-132 did not have any detectable free SN-38 or SN-38G, butinstead, all SN-38 in the tumor was bound to IMMU-132. Importantly,since no SN-38G was detected in the tumors, this suggests SN-38 bound toIMMU-132 was not glucuronidated. The AUC for SN-38 bound to IMMU-132 inthese tumors was 54.3 μg/g·h, which is 135-fold higher than the amountof SN-38 in the tumors of animals treated with irinotecan over the 2-hperiod that SN-38 could be detected, even though mice given irinotecanreceived 28-fold more SN-38 equivalents than administered with IMMU-132(i.e., 448 vs 16 μg SN-38 equivalents, respectively)

Conclusions

IMMU-132 is a humanized anti-Trop-2 antibody conjugated with 7.6molecules of SN-38, the active metabolite of irinotecan, a topoisomeraseI inhibitor. Clinically, IMMU-132 has shown manageable toxicity andencouraging responses in patients with relapsed/refractory TNBC(ClinicalTrials.gov, NCT01631552). IMMU-132 therapy alone demonstratedsignificant anti-tumor effects in human TNBC xenografts at a humanequivalent dose that is 5-fold less than that being used clinically(i.e., 10 mg/kg). Since preclinical studies indicate IMMU-132 can becombined with two different microtubule-inhibitors or a PARP-inhibitorwith significantly enhanced anti-tumor activity, these data support theuse of IMMU-132 and other antibody-drug conjugates (ADCs) that cause DNAbreaks, in combination with microtubule inhibitors and/or PARPinhibitors in general, as well as other chemotherapeutic agents thattarget cell division through microtubule inhibition or DNA-repairmechanisms. A preferred ADC class is represented by anti-Trop-2 antibodyconjugates in patients with Trop-2 positive cancers, including but notlimited to TNBC, metastatic colon cancer, SCLC and NSCLC, since this isa target that is expressed in high amounts in a large number of cancers,and is localized on the cell surface and cytoplasmically in the cancercells. However, other cancer antigen targets can also be used for theADC in this application if they bear a drug that also causes DNA breaks,such as via targeting topoisomerase I, like SN-38.

Synergy was achieved when IMMU-132 was combined with PARP-inhibitors(e.g., olaparib) in TNBC tumor lines that had BRCA1/2 defects, as wellas wild-type expression, including one with only a PTEN defect. Thissuggests that IMMU-132 may synergize with any tumor that has any kind ofdisruption in DNA homologous recombination pathways. Combined witholaparib, IMMU-132 therapy achieved significant anti-tumor effects abovethat observed with monotherapy with each, resulting in a significantsurvival benefit. IMMU-132 combined with microtubule inhibitors, (e.g.,paclitaxel or eribulin mesylate) also enhanced efficacy significantlycompared to monotherapy with each agent.

Overall, these data evidence the unexpected significant advantage ofcombination therapy with an antibody-drug conjugate (ADC) that targetscancer cells and induces DNA strand breaks, such as IMMU-132, andmicrotubule inhibitors or PARP inhibitors. Targeting the PARP DNA repairpathway in BRCA1/2 mutant TNBC tumors by combining IMMU-132 therapy witheither paclitaxel or olaparib achieved synthetic lethality in thisdisease model with no observable toxicity. In an exemplary embodiment,the combination of IMMU-132 and a PARP or microtubule inhibitor is ofuse to treat Trop-2 positive cancers, such as TNBC. These data providethe rationale for use of IMMU-132 in combination with otherchemotherapeutics that likewise target DNA-repair mechanisms in patientswith TNBC or similar tumors.

Example 2 Combination Therapy with IMMU-114 (Anti-HLA-DR) Plus BrutonKinase Inhibitor in Chronic Lymphocytic Leukemia (CLL)

Experimental Design.

Human chronic B cell leukemia cells (JVM-3) were plated in a 96-wellplate. Cells were then incubated with various doses of the Bruton kinaseinhibitor ibrutinib (1×10⁻⁵ to 3.9×10⁻⁹M) plus a constant amount ofIMMU-114 (0.25, 0.5, 0.75, or 1 nM) for 96 h. Cell viability wasassessed by MTS assay and dose/response curves were generated based onpercent growth inhibition relative to untreated control cells.IC₅₀-values were determined using Prism Graph-Pad. Data were normalizedand isobolograms generated to demonstrate the effect of IMMU-114 onibrutinib in JVM-3 cells.

Results

An example of how IMMU-114 shifted the IC₅₀ of ibrutinib is shown inFIG. 5A. Cells incubated with ibrutinib alone resulted in an IC₅₀ of2.2×10⁻⁶M. However, when 0.5 nM of IMMU-114 is combined with ibrutinib,the IC₅₀-value drops 2-fold to 1.08×10⁻⁶M. It should be noted thatIMMU-114 at 0.5 nM only results in an approximate 22% inhibition ofgrowth. Likewise, 0.75 nM IMMU-114 shifted ibrutinib IC₅₀-value downward5.7-fold.

These data, along with the results from two other assays, werenormalized to produce an isobologram in order to ascertain whether thisinteraction was synergistic, additive, or antagonistic (FIG. 6A). Asshown, when the JVM-3 cells were incubated with ibrutinib combined withIMMU-114, an additive effect is evident. These data demonstrate thatwhile each agent alone is capable of inhibiting the growth of CLL invitro, when combined an additive effect is achieved which supportscombining IMMU-114 and Bruton kinase therapies in patients with CLL.

Example 3 Combination Therapy with IMMU-114 (Anti-HLA-DR) PlusPhosphatidylinositide 3-Kinase (PI3K) Inhibitor in Chronic LymphocyticLeukemia (CLL)

Experimental Design.

Human chronic leukemia B cells (JVM-3) were plated in a 96-well plate.Cells were then incubated with various doses of a PI3K inhibitoridelalisib (1×10⁻⁵ to 3.9×10⁻⁹M) plus a constant amount of IMMU-114(0.25, 0.5, 0.75, or 1 nM) for 96 h. Cell viability was assessed by MTSassay and dose/response curves were generated based on percent growthinhibition relative to untreated control cells. IC₅₀-values weredetermined using Prism Graph-Pad. Data were normalized and isobologramsgenerated to demonstrate the effect of IMMU-114 on ibrutinib in JVM-3cells.

Results

As with ibrutinib, IMMU-114 shifted the IC₅₀ of idelalisib in JVM-3cells (FIG. 5B). Cells incubated with idelalisib alone resulted in anIC₅₀ of 1.51×10⁻⁶M. However, when 0.5 nM of IMMU-114 is combined withidelalisib, the IC₅₀-value drops 2-fold to 0.77×10⁻⁶M (IMMU-114 at 0.5nM only results in an approximate 26% inhibition of growth). Likewise, 1nM IMMU-114 shifted ibrutinib IC₅₀-value downward >18-fold.

These data, along with the results from one other assay, were normalizedto produce an isobologram (FIG. 6B). As shown, an additive effect wasachieved when JVM-3 cells were incubated with idelalisib combined withIMMU-114. These data demonstrate the utility of combining IMMU-114 witha PI3K inhibitor to treat CLL clinically.

Example 4 Treatment of Relapsed Chronic Lymphocytic Leukemia withIMMU-114 and Ibrutinib

A 65-year-old woman with a history of CLL, as defined by theInternational Workshop on Chronic Lymphocytic Leukemia and World HealthOrganization classifications, presents with relapsed disease after priortherapies with fludarabine, dexamethasone, and rituximab, as well as aregimen of CVP. She now has fever and night sweats associated withgeneralized lymph node enlargement, a reduced hemoglobin and plateletproduction, as well as a rapidly rising leukocyte count. Her LDH iselevated and the beta-2-microglobulin is almost twice normal. Thepatient is given therapy with anti-HLA-DR IMMU-114 (IgG4 hL243) at 200mg administered subcutaneously twice weekly. The antibody is given incombination with the Bruton kinase inhibitor ibrutinib, administeredorally at a dosage of 420 mg daily. After 6 weeks, evaluation of thepatient's hematological and lab values indicate that she has shown apartial response to the combination therapy.

Example 5 Treatment of Relapsed/Refractory Non-Hodgkin's Lymphoma (NHL)with IMMU-114 and Idelalisib

Seventeen patients with follicular NHL who have not had a response torituximab and an alkylating agent, or have had a relapse within 6 monthsafter receipt of those therapies receive 200 mg IMMU-114, injected s.c.twice weekly, in combination with the PI3K inhibitor idelalisib,administered orally at 150 mg twice daily, until the disease progressesor the patient withdraws from the study. Responses are assessed by CTscans, with other evaluations including adverse events, B-cell bloodlevels, serum IMMU-114 levels and human anti-IMMU-114 (HAHA) titers.

Only occasional, mild to moderate transient injection reactions are seenand no other safety issues except neutropenia up to Grade 3 (butreversible after interrupting therapy until reduced to Grade 1) areobserved. Transient B-cell depletion (up to about 25%) is observed. Theobjective response rate (partial responses plus complete responses pluscomplete responses unconfirmed) is 47% ( 8/17) with a completeresponse/complete response unconfirmed rate of 24% ( 4/17). Four of theeight objective responses continue for 30 weeks or more. All serumsamples evaluated for human anti-IMMU-114 antibody (HAHA) are negative.

Example 6 Conjugation of Bifunctional SN-38 Products to Mildly ReducedAntibodies

The anti-CEACAM5 humanized MAb, hMN-14 (also known as labetuzumab), theanti-CD22 humanized MAb, hLL2 (also known as epratuzumab), the anti-CD20humanized MAb, hA20 (also known as veltuzumab), the anti-EGP-1 humanizedMAb, hRS7, and anti-mucin humanized MAb, hPAM4 (also known asclivatuzumab), were used in these studies. Each antibody was reducedwith dithiothreitol (DTT), used in a 50-to-70-fold molar excess, in 40mM PBS, pH 7.4, containing 5.4 mM EDTA, at 37° C. (bath) for 45 min. Thereduced product was purified by size-exclusion chromatography and/ordiafiltration, and was buffer-exchanged into a suitable buffer at pH6.5. The thiol content was determined by Ellman's assay, and was in the6.5-to-8.5 SH/IgG range. Alternatively, the antibodies were reduced withTris (2-carboxyethyl) phosphine (TCEP) in phosphate buffer at pH in therange of 5-7, followed by in situ conjugation. The reduced MAb wasreacted with ˜10-to-15-fold molar excess of CL2A-SN-38 (prepared asdisclosed in U.S. Pat. No. 7,999,083) using DMSO at 7-15% v/v asco-solvent, and incubating for 20 min at ambient temperature. Theconjugate was purified by centrifuged SEC, passage through a hydrophobiccolumn, and finally by ultrafiltration-diafiltration. The product wasassayed for SN-38 by absorbance at 366 nm and correlating with standardvalues, while the protein concentration was deduced from absorbance at280 nm, corrected for spillover of SN-38 absorbance at this wavelength.This way, the SN-38/MAb substitution ratios were determined. Thepurified conjugates were stored as lyophilized formulations in glassvials, capped under vacuum and stored in a −20° C. freezer. SN-38 molarsubstitution ratios (MSR) obtained for some of these conjugates, whichwere typically in the 5-to-7 range, are shown in Table 10. The person ofordinary skill will realize that the conjugation method may be appliedto any antibody of use in the disclosed methods.

TABLE 10 SN-38/MAb Molar substitution ratios (MSR) in some conjugatesMAb Conjugate MSR hMN-14 hMN-14-CL2A-SN-38 6.1 hRS7 hRS7-CL2A-SN-38 5.8hA20 hA20-CL2A-SN-38 5.8 hLL2 hLL2-CL2A-SN-38 5.7 hPAM4 hPAM4-CL2A-SN-385.9

Example 7 In Vivo Therapeutic Efficacies in Preclinical Models of HumanPancreatic or Colon Carcinoma

Immune-compromised athymic nude mice (female), bearing subcutaneoushuman pancreatic or colon tumor xenografts were treated with eitherspecific CL2A-SN-38 conjugate or control conjugate or were leftuntreated. The therapeutic efficacies of the specific conjugates wereobserved. FIG. 7 shows a Capan 1 pancreatic tumor model, whereinspecific CL2A-SN-38 conjugates of hRS7 (anti-EGP-1), hPAM4 (anti-mucin),and hMN-14 (anti-CEACAM5) antibodies showed better efficacies thancontrol hA20-CL2A-SN-38 conjugate (anti-CD20) and untreated control.Similarly in a BXPC3 model of human pancreatic cancer, the specifichRS7-CL2A-SN-38 showed better therapeutic efficacy than controltreatments (FIG. 8). Likewise, in an aggressive LS174T model of humancolon carcinoma, treatment with specific hMN-14-CL2A-SN-38 was moreefficacious than non-treatment (FIG. 9).

Example 8 In Vivo Therapy of Lung Metastases of GW-39 Human ColonicTumors in Nude Mice Using hMN-14-[CL1-SN-38] and hMN-14-[CL2-SN-38]

A lung metastatic model of colonic carcinoma was established in nudemice by i.v. injection of GW-39 human colonic tumor suspension, andtherapy was initiated 14 days later. Specific anti-CEACAM5 antibodyconjugates, hMN14-CL1-SN-38 and hMN14-CL2-SN-38, as well as nontargetinganti-CD22 MAb control conjugates, hLL2-CL1-SN-38 and hLL2-CL2-SN-38 andequidose mixtures of hMN14 and SN-38 were injected at a dose schedule ofq4dx8, using different doses. FIG. 10 (MSR=SN-38/antibody molarsubstitution ratio) shows selective therapeutic effects due to hMN-14conjugates. At equivalent dosages of 250 μg, the mice treated withhMN14-CL1-SN-38 or hMN14-CL2-SN-38 showed a median survival of greaterthan 107 days. Mice treated with the control conjugated antibodieshLL2-CL1-SN-38 and hLL2-CL2-SN-38, which do not specifically target lungcancer cells, showed median survival of 56 and 77 days, while micetreated with unconjugated hMN14 IgG and free SN-38 showed a mediansurvival of 45 days, comparable to the untreated saline control of 43.5days. A significant and surprising increase in effectiveness of theconjugated, cancer cell targeted antibody-SN-38 conjugate, which wassubstantially more effective than unconjugated antibody and freechemotherapeutic agent alone, was clearly seen. The dose-responsivenessof therapeutic effect of conjugated antibody was also observed. Theseresults demonstrate the clear superiority of the SN-38-antibodyconjugates compared to the combined effect of both unconjugated antibodyand free SN-38 in the same in vivo human lung cancer system.

Example 9 Use of Humanized Anti-Trop-2 IgG-SN-38 Conjugate for EffectiveTreatment of Diverse Epithelial Cancers

Summary

The purpose of this study was to evaluate the efficacy of anSN-38-anti-Trop-2 antibody-drug conjugate (ADC) against several humansolid tumor types, and to assess its tolerability in mice and monkeys,the latter with tissue cross-reactivity to hRS7 similar to humans. TwoSN-38 derivatives, CL2-SN-38 and CL2A-SN-38, were conjugated to theanti-Trop-2-humanized antibody, hRS7. The immunoconjugates werecharacterized in vitro for stability, binding, and cytotoxicity.Efficacy was tested in five different human solid tumor-xenograft modelsthat expressed Trop-2 antigen. Toxicity was assessed in mice and inCynomolgus monkeys.

The hRS7 conjugates of the two SN-38 derivatives were equivalent in drugsubstitution (˜6), cell binding (K_(d)˜1.2 nmol/L), cytotoxicity(IC₅₀˜2.2 nmol/L), and serum stability in vitro (t/_(1/2)˜20 hours).Exposure of cells to the ADC demonstrated signaling pathways leading toPARP cleavage, but differences versus free SN-38 in p53 and p21upregulation were noted. Significant antitumor effects were produced byhRS7-SN-38 at nontoxic doses in mice bearing Calu-3 (P≤0.05), Capan-1(P<0.018), BxPC-3 (P<0.005), and COLO 205 tumors (P<0.033) when comparedto nontargeting control ADCs. Mice tolerated a dose of 2×12 mg/kg (SN-38equivalents) with only short-lived elevations in ALT and AST liverenzyme levels. Cynomolgus monkeys infused with 2×0.96 mg/kg exhibitedonly transient decreases in blood counts, although, importantly, thevalues did not fall below normal ranges.

We conclude that the anti-Trop-2 hRS7-CL2A-SN-38 ADC providedsignificant and specific antitumor effects against a range of humansolid tumor types. It was well tolerated in monkeys, with tissue Trop-2expression similar to humans. (Cardillo et al., 2011, Clin Cancer Res17:3157-69.)

Successful irinotecan treatment of patients with solid tumors has beenlimited due in large part to the low conversion rate of the CPT-11prodrug into the active SN-38 metabolite. Others have examinednontargeted forms of SN-38 as a means to bypass the need for thisconversion and to deliver SN-38 passively to tumors. We conjugated SN-38covalently to a humanized anti-Trop-2 antibody, hRS7. This antibody-drugconjugate has specific antitumor effects in a range of s.c. human cancerxenograft models, including non-small cell lung carcinoma, pancreatic,colorectal, and squamous cell lung carcinomas, all at nontoxic doses(e.g., ≤3.2 mg/kg cumulative SN-38 equivalent dose).

Trop-2 is widely expressed in many epithelial cancers, but also somenormal tissues, and therefore a dose escalation study in Cynomolgusmonkeys was performed to assess the clinical safety of this conjugate.Monkeys tolerated 24 mg SN-38 equivalents/kg with only minor,reversible, toxicities. Given its tumor-targeting and safety profile,hRS7-SN-38 may provide an improvement in the management of solid tumorsresponsive to irinotecan.

Introduction

Human trophoblast cell-surface antigen (Trop-2), also known as GA733-1(gastric antigen 733-1), EGP-1 (epithelial glycoprotein-1), and TACSTD2(tumor-associated calcium signal transducer), is expressed in a varietyof human carcinomas and has prognostic significance in some, beingassociated with more aggressive disease (see, e.g., Alberti et al.,1992, Hybridoma 11:539-45; Stein et al., 1993, Int J Cancer 55:938-46;Stein et al., 1994, Int J Cancer Suppl. 8:98-102). Studies of thefunctional role of Trop-2 in a mouse pancreatic cancer cell linetransfected with murine Trop-2 revealed increased proliferation in lowserum conditions, migration, and anchorage-independent growth in vitro,and enhanced growth rate with evidence of increased Ki-67 expression invivo and a higher likelihood to metastasize (Cubas et al., 2010, MolCancer 9:253).

Trop-2 antigen's distribution in many epithelial cancers makes it anattractive therapeutic target. Stein and colleagues (1993, Int J Cancer55:938-46) characterized an antibody, designated RS7-3G11 (RS7), thatbound to EGP-1, which was present in a number of solid tumors, but theantigen was also expressed in some normal tissues, usually in a lowerintensity, or in restricted regions. Targeting and therapeuticefficacies were documented in a number of human tumor xenografts usingradiolabeled RS7 (Shih et al., 1995, Cancer Res 55:5857s-63s; Stein etal., 1997, Cancer 80:2636-41; Govindan et al., 2004, Breast Cancer ResTreat 84:173-82), but this internalizing antibody did not showtherapeutic activity in unconjugated form (Shih et al., 1995, Cancer Res55:5857s-63s). However, in vitro it has demonstrated antibody-dependentcellular cytotoxicity (ADCC) activity against Trop-2 positivecarcinomas.

We reported the preparation of antibody-drug conjugates (ADC) using ananti-CEACAM5 (CD66e) IgG coupled to several derivatives of SN-38, atopoisomerase-I inhibitor that is the active component of irinotecan, orCPT-11 (Moon et al., 2008, J Med Chem 51:6916-26; Govindan et al., 2009,Clin Cancer Res 15:6052-61). The derivatives varied in their in vitroserum stability properties, and in vivo studies found one form(designated CL2) to be more effective in preventing or arresting thegrowth of human colonic and pancreatic cancer xenografts than otherlinkages with more or less stability.

Importantly, these effects occurred at nontoxic doses, with initialtesting failing to determine a dose-limiting toxicity (Govindan et al.,2009, Clin Cancer Res 15:6052-61). These results were encouraging, butalso surprising, because the CEACAM5 antibody does not internalize, aproperty thought to be critical to the success of an ADC. We speculatedthat the therapeutic activity of the anti-CEACAM5-SN-38 conjugate mightbe related to the slow release of SN-38 within the tumor after theantibody localized. Because irinotecan performs best when cells areexposed during the S-phase of their growth cycle, a sustained release isexpected to improve responses. Indeed, SN-38 coupled to nontargeting,plasma extending agents, such as polyethylene glycol (PEG) or micelles,has shown improved efficacy over irinotecan or SN-38 alone (e.g.,Koizumi et al., 2006, Cancer Res 66:10048-56), lending additionalsupport to this mechanism.

Given the RS7 antibody's broad reactivity with epithelial cancers andits internalization ability, we hypothesized that an RS7-SN-38 conjugatecould benefit not only from the sustained release of the drug, but alsofrom direct intracellular delivery. Therefore, we prepared and testedthe efficacy of SN-38 conjugates using a humanized version of the murineRS7 antibody (hRS7), wherein the SN-38 was attached to the antibodyusing an improved CL2A linker, which improved the quality of theconjugate and its in vivo efficacy without altering its in vitrostability. This new derivative (designated CL2A) is a preferred agentfor SN-38 coupling to antibodies.

Herein, we show the efficacy of the hRS7-SN-38 conjugate in severalepithelial cancer cell lines implanted in nude mice at nontoxic dosages,with other studies revealing that substantially higher doses could betolerated. More importantly, toxicity studies in monkeys that alsoexpress Trop-2 in similar tissues as humans showed that hRS7-SN-38 wastolerated at appreciably higher amounts than the therapeuticallyeffective dose in mice.

Materials and Methods

Cell lines, antibodies, and chemotherapeutics. All human cancer celllines used in this study were purchased from the American Type CultureCollection. These include Calu-3 (non-small cell lung carcinoma),SK-MES-1 (squamous cell lung carcinoma), COLO 205 (colonicadenocarcinoma), Capan-1 and BxPC-3 (pancreatic adenocarcinomas), andPC-3 (prostatic adenocarcinomas). Humanized RS7 IgG and controlhumanized anti-CD20 (hA20 IgG, veltuzumab) and anti-CD22 (hLL2 IgG,epratuzumab) antibodies were prepared at Immunomedics, Inc. Irinotecan(20 mg/mL) was obtained from Hospira, Inc.

SN-38 immunoconjugates and in vitro aspects. Synthesis of CL2-SN-38 hasbeen described previously (Moon et al., 2008, J Med Chem 51:6916-26).Its conjugation to hRS7 IgG and serum stability were performed asdescribed (Moon et al., 2008, J Med Chem 51:6916-26; Govindan et al.,2009, Clin Cancer Res 15:6052-61). Preparations of CL2A-SN-38 (M.W.1480) and its hRS7 conjugate, and stability, binding, and cytotoxicitystudies, were conducted as described previously (Moon et al., 2008, JMed Chem 51:6916-26). Cell lysates were prepared and immunoblotting forp21^(Waf1/Cip), p53, and PARP (poly-ADP-ribose polymerase) wasperformed.

In vivo therapeutic studies. For all animal studies, the doses of SN-38immunoconjugates and irinotecan are shown in SN-38 equivalents. Based ona mean SN-38/IgG substitution ratio of 6, a dose of 500 μg ADC to a 20-gmouse (25 mg/kg) contains 0.4 mg/kg of SN-38. Irinotecan doses arelikewise shown as SN-38 equivalents (i.e., 40 mg irinotecan/kg isequivalent to 24 mg/kg of SN-38). NCr female athymic nude (nu/nu) mice,4 to 8 weeks old, and male Swiss-Webster mice, 10 weeks old, werepurchased from Taconic Farms. Tolerability studies were performed inCynomolgus monkeys (Macaca fascicularis; 2.5-4 kg male and female) bySNBL USA, Ltd. Animals were implanted subcutaneously with differenthuman cancer cell lines. Tumor volume (TV) was determined bymeasurements in 2 dimensions using calipers, with volumes defined as:L×w²/2, where L is the longest dimension of the tumor and w is theshortest. Tumors ranged in size between 0.10 and 0.47 cm³ when therapybegan. Treatment regimens, dosages, and number of animals in eachexperiment are described in the Results. The lyophilized hRS7-CL2A-SN-38and control ADC were reconstituted and diluted as required in sterilesaline. All reagents were administered intraperitoneally (0.1 mL),except irinotecan, which was administered intravenously. The dosingregimen was influenced by our prior investigations, where the ADC wasgiven every 4 days or twice weekly for varying lengths of time (Moon etal., 2008, J Med Chem 51:6916-26; Govindan et al., 2009, Clin Cancer Res15:6052-61). This dosing frequency reflected a consideration of theconjugate's serum half-life in vitro, to allow a more continuousexposure to the ADC.

Statistics. Growth curves were determined as percent change in initialTV over time. Statistical analysis of tumor growth was based on areaunder the curve (AUC). Profiles of individual tumor growth were obtainedthrough linear-curve modeling. An f-test was employed to determineequality of variance between groups before statistical analysis ofgrowth curves. A 2-tailed t-test was used to assess statisticalsignificance between the various treatment groups and controls, exceptfor the saline control, where a 1-tailed t-test was used (significanceat P≤0.05). Statistical comparisons of AUC were performed only up to thetime that the first animal within a group was euthanized due toprogression.

Pharmacokinetics and biodistribution. ¹¹¹In-radiolabeled hRS7-CL2A-SN-38and hRS7 IgG were injected into nude mice bearing s.c. SK-MES-1 tumors(˜0.3 cm³). One group was injected intravenously with 20 μCi (250-μgprotein) of ¹¹¹In-hRS7-CL2A-SN-38, whereas another group received 20 μCi(250-μg protein) of ¹¹¹In-hRS7 IgG. At various timepoints mice (5 pertimepoint) were anesthetized, bled via intracardiac puncture, and theneuthanized. Tumors and various tissues were removed, weighed, andcounted by y scintillation to determine the percentage injected dose pergram tissue (% ID/g). A third group was injected with 250 μg ofunlabeled hRS7-CL2A-SN-38 3 days before the administration of¹¹¹In-hRS7-CL2A-SN-38 and likewise necropsied. A 2-tailed t-test wasused to compare hRS7-CL2A-SN-38 and hRS7 IgG uptake after determiningequality of variance using the f-test. Pharmacokinetic analysis on bloodclearance was performed using WinNonLin software (Parsight Corp.).

Tolerability in Swiss-Webster mice and Cynomolgus monkeys. Briefly, micewere sorted into 4 groups each to receive 2-mL i.p. injections of eithera sodium acetate buffer control or 3 different doses of hRS7-CL2A-SN-38(4, 8, or 12 mg/kg of SN-38) on days 0 and 3 followed by blood and serumcollection, as described in Results. Cynomolgus monkeys (3 male and 3female; 2.5-4.0 kg) were administered 2 different doses ofhRS7-CL2A-SN-38. Dosages, times, and number of monkeys bled forevaluation of possible hematologic toxicities and serum chemistries aredescribed in the Results.

Results

Stability and potency of hRS7-CL2A-SN-38. Two different linkages wereused to conjugate SN-38 to hRS7 IgG. The first is termed CL2-SN-38 andhas been described previously (Moon et al., 2008, J Med Chem 51:6916-26;Govindan et al., 2009, Clin Cancer Res 15:6052-61). A change was made tothe synthesis of the CL2 linker in that the phenylalanine moiety wasremoved. This change simplified the synthesis, but did not affect theconjugation outcome (e.g., both CL2-SN-38 and CL2A-SN-38 incorporated ˜6SN-38 per IgG molecule). Side-by-side comparisons found no significantdifferences in serum stability, antigen binding, or in vitrocytotoxicity (not shown).

To confirm that the change in the SN-38 linker from CL2 to CL2A did notimpact in vivo potency, hRS7-CL2A and hRS7-CL2-SN-38 were compared inmice bearing COLO 205 or Capan-1 tumors (not shown), using 0.4 mg or 0.2mg/kg SN-38 twice weekly×4 weeks, respectively, and with starting tumorsof 0.25 cm³ size in both studies. Both the hRS7-CL2A and CL2-SN-38conjugates significantly inhibited tumor growth compared to untreated(AUC_(14days)P<0.002 vs. saline in COLO 205 model; AUC_(21days)P<0.001vs. saline in Capan-1 model), and a nontargeting anti-CD20 control ADC,hA20-CL2A-SN-38 (AUC_(14days)P<0.003 in COLO-205 model; AUC_(35days):P<0.002 in Capan-1 model). At the end of the study (day 140) in theCapan-1 model, 50% of the mice treated with hRS7-CL2A-SN-38 and 40% ofthe hRS7-CL2-SN-38 mice were tumor-free, whereas only 20% of thehA20-ADC-treated animals had no visible sign of disease.

Mechanism of action. In vitro cytotoxicity studies demonstrated thathRS7-CL2A-SN-38 had IC₅₀ values in the nmol/L range against severaldifferent solid tumor lines (Table 11). The IC₅₀ with free SN-38 waslower than the conjugate in all cell lines. Although there was nocorrelation between Trop-2 expression and sensitivity tohRS7-CL2A-SN-38, the IC₅₀ ratio of the ADC versus free SN-38 was lowerin the higher Trop-2-expressing cells, most likely reflecting theenhanced ability to internalize the drug when more antigen is present.

TABLE 11 Expression of Trop-2 and in vitro cytotoxicity of SN-38 andhRS7-SN-38 in several solid tumor lines Cytotoxicity results Trop-2expression via FACS hRS7-SN- Median SN-38 95% CI 38 ^(a) 95% CI Cellfluorescence Percent IC₅₀ IC₅₀ IC₅₀ IC₅₀ ADC/free line (background)positive (nmol/L) (nmol/L) (nmol/L) (nmol/L) SN-38 ratio Calu-3 282.2(4.7) 99.6% 7.19 5.77-8.95 9.97  8.12-12.25 1.39 COLO 141.5 (4.5) 99.5%1.02 0.66-1.57 1.95  1.26-3.01 1.91 205 Capan-1 100.0 (5.0) 94.2% 3.502.17-5.65 6.99  5.02-9.72 2.00 PC-3 46.2 (5.5) 73.6% 1.86 1.16-2.99 4.24 2.99-6.01 2.28 SK- 44.0 (3.5) 91.2% 8.61 6.30-11.76 23.14 17.98-29.782.69 MES-1 BxPC-3 26.4 (3.1) 98.3% 1.44 1.04-2.00 4.03  3.25-4.98 2.80^(a)IC₅₀-value is shown as SN-38 equivalents of hRS7-SN-38

SN-38 is known to activate several signaling pathways in cells, leadingto apoptosis. Our initial studies examined the expression of 2 proteinsinvolved in early signaling events (p21^(Waf1/Cip1) and p53) and 1 lateapoptotic event [cleavage of poly-ADP-ribose polymerase (PARP)] in vitro(not shown). In BxPC-3, SN-38 led to a 20-fold increase inp21^(Waf1/Cip1) expression, whereas hRS7-CL2A-SN-38 resulted in only a10-fold increase, a finding consistent with the higher activity withfree SN-38 in this cell line (Table 11). However, hRS7-CL2A-SN-38increased p21^(Waf1/Cip1) expression in Calu-3 more than 2-fold overfree SN-38 (not shown).

A greater disparity between hRS7-CL2A-SN-38- and free SN-38-mediatedsignaling events was observed in p53 expression. In both BxPC-3 andCalu-3, upregulation of p53 with free SN-38 was not evident until 48hours, whereas hRS7-CL2A-SN-38 upregulated p53 within 24 hours (notshown). In addition, p53 expression in cells exposed to the ADC washigher in both cell lines compared to SN-38 (not shown). Interestingly,although hRS7 IgG had no appreciable effect on p21^(Waf1/Cip1)expression, it did induce the upregulation of p53 in both BxPC-3 andCalu-3, but only after a 48-hour exposure. In terms of later apoptoticevents, cleavage of PARP was evident in both cell lines when incubatedwith either SN-38 or the conjugate (not shown). The presence of thecleaved PARP was higher at 24 hours in BxPC-3, which correlates withhigh expression of p21 and its lower IC₅₀. The higher degree of cleavagewith free SN-38 over the ADC was consistent with the cytotoxicityfindings.

Efficacy of hRS7-SN-38. Because Trop-2 is widely expressed in severalhuman carcinomas, studies were performed in several different humancancer models, which started with an evaluation of the hRS7-CL2-SN-38linkage, but later, conjugates with the CL2A-linkage were used.Calu-3-bearing nude mice given 0.04 mg SN-38/kg of the hRS7-CL2-SN-38every 4 days×4 had a significantly improved response compared to animalsadministered the equivalent amount of hLL2-CL2-SN-38 (TV=0.14±0.22 cm³vs. 0.80±0.91 cm³, respectively; AUC_(42days)P<0.026; FIG. 11A). Adose-response was observed when the dose was increased to 0.4 mg/kgSN-38. At this higher dose level, all mice given the specific hRS7conjugate were “cured” within 28 days, and remained tumor-free until theend of the study on day 147, whereas tumors regrew in animals treatedwith the irrelevant ADC (specific vs. irrelevant AUC_(98days): P=0.05).In mice receiving the mixture of hRS7 IgG and SN-38, tumorsprogressed >4.5-fold by day 56 (TV=1.10±0.88 cm³; AUC_(56days)P<0.006vs. hRS7-CL2-SN-38).

Efficacy also was examined in human colonic (COLO 205) and pancreatic(Capan-1) tumor xenografts. In COLO 205 tumor-bearing animals, (FIG.11B), hRS7-CL2-SN-38 (0.4 mg/kg, q4dx8) prevented tumor growth over the28-day treatment period with significantly smaller tumors compared tocontrol anti-CD20 ADC (hA20-CL2-SN-38), or hRS7 IgG (TV=0.16±0.09 cm³,1.19±0.59 cm³, and 1.77±0.93 cm³, respectively; AUC_(28days)P<0.016).The MTD of irinotecan (24 mg SN-38/kg, q2dx5) was as effective ashRS7-CL2-SN-38, because mouse serum can more efficiently convertirinotecan to SN-38 than human serum, but the SN-38 dose in irinotecan(2,400 μg cumulative) was 37.5-fold greater than with the conjugate (64μg total).

Animals bearing Capan-1 showed no significant response to irinotecanalone when given at an SN-38-dose equivalent to the hRS7-CL2-SN-38conjugate (e.g., on day 35, average tumor size was 0.04±0.05 cm³ inanimals given 0.4 mg SN-38/kg hRS7-SN-38 vs. 1.78±0.62 cm³ inirinotecan-treated animals given 0.4 mg/kg SN-38; AUC_(day35)P<0.001;FIG. 11C). When the irinotecan dose was increased 10-fold to 4 mg/kgSN-38, the response improved, but still was not as significant as theconjugate at the 0.4 mg/kg SN-38 dose level (TV=0.17±0.18 cm³ vs.1.69±0.47 cm³, AUC_(day49)P<0.001). An equal dose of nontargetinghA20-CL2-SN-38 also had a significant antitumor effect as compared toirinotecan-treated animals, but the specific hRS7 conjugate wassignificantly better than the irrelevant ADC (TV=0.17±0.18 cm³ vs.0.80±0.68 cm³, AUC_(day49)P<0.018).

Studies with the hRS7-CL2A-SN-38 ADC were then extended to 2 othermodels of human epithelial cancers. In mice bearing BxPC-3 humanpancreatic tumors (FIG. 11D), hRS7-CL2A-SN-38 again significantlyinhibited tumor growth in comparison to control mice treated with salineor an equivalent amount of nontargeting hA20-CL2A-SN-38 (TV=0.24±0.11cm³ vs. 1.17±0.45 cm³ and 1.05±0.73 cm³, respectively;AUC_(day)21P<0.001), or irinotecan given at a 10-fold higher SN-38equivalent dose (TV=0.27±0.18 cm³ vs. 0.90±0.62 cm³, respectively;AUC_(day)25P<0.004). Interestingly, in mice bearing SK-MES-1 humansquamous cell lung tumors treated with 0.4 mg/kg of the ADC (FIG. 11E),tumor growth inhibition was superior to saline or unconjugated hRS7 IgG(TV=0.36±0.25 cm³ vs. 1.02±0.70 cm³ and 1.30±1.08 cm³, respectively;AUC_(28 days), P<0.043), but nontargeting hA20-CL2A-SN-38 or the MTD ofirinotecan provided the same antitumor effects as the specifichRS7-SN-38 conjugate. In all murine studies, the hRS7-SN-38 ADC was welltolerated in terms of body weight loss (not shown).

Biodistribution of hRS7-CL2A-SN-38. The biodistributions ofhRS7-CL2A-SN-38 or unconjugated hRS7 IgG were compared in mice bearingSK-MES-1 human squamous cell lung carcinoma xenografts (not shown),using the respective ¹¹¹In-labeled substrates. A pharmacokineticanalysis was performed to determine the clearance of hRS7-CL2A-SN-38relative to unconjugated hRS7 (not shown). The ADC cleared faster thanthe equivalent amount of unconjugated hRS7, with the ADC exhibiting ˜40%shorter half-life and mean residence time. Nonetheless, this had aminimal impact on tumor uptake (not shown). Although there weresignificant differences at the 24- and 48-hour timepoints, by 72 hours(peak uptake) the amounts of both agents in the tumor were similar.Among the normal tissues, hepatic and splenic differences were the moststriking (not shown). At 24 hours postinjection, there was >2-fold morehRS7-CL2A-SN-38 in the liver than hRS7 IgG. Conversely, in the spleenthere was 3-fold more parental hRS7 IgG present at peak uptake (48-hourtimepoint) than hRS7-CL2A-SN-38. Uptake and clearance in the rest of thetissues generally reflected differences in the blood concentration.

Because twice-weekly doses were given for therapy, tumor uptake in agroup of animals that first received a predose of 0.2 mg/kg (250 μgprotein) of the hRS7 ADC 3 days before the injection of the¹¹¹In-labeled antibody was examined. Tumor uptake of¹¹¹In-hRS7-CL2A-SN-38 in predosed mice was substantially reduced atevery timepoint in comparison to animals that did not receive thepredose (e.g., at 72 hours, predosed tumor uptake was 12.5%±3.8% ID/gvs. 25.4%±8.1% ID/g in animals not given the predose; P=0.0123).Predosing had no appreciable impact on blood clearance or tissue uptake(not shown). These studies suggest that in some tumor models, tumoraccretion of the specific antibody can be reduced by the precedingdose(s), which likely explains why the specificity of a therapeuticresponse could be diminished with increasing ADC doses and why furtherdose escalation is not indicated.

Tolerability of hRS7-CL2A-SN-38 in Swiss-Webster mice and Cynomolgusmonkeys. Swiss-Webster mice tolerated 2 doses over 3 days, each of 4, 8,and 12 mg SN-38/kg of the hRS7-CL2A-SN-38, with minimal transient weightloss (not shown). No hematopoietic toxicity occurred and serumchemistries only revealed elevated aspartate transaminase (AST) andalanine transaminase (not shown). Seven days after treatment, AST roseabove normal levels (>298 U/L) in all 3 treatment groups (not shown),with the largest proportion of mice being in the 2×8 mg/kg group.However, by 15 days posttreatment, most animals were within the normalrange. ALT levels were also above the normal range (>77 U/L) within 7days of treatment (not shown) and with evidence of normalization by Day15. Livers from all these mice did not show histologic evidence oftissue damage (not shown). In terms of renal function, only glucose andchloride levels were somewhat elevated in the treated groups. At 2×8mg/kg, 5 of 7 mice had slightly elevated glucose levels (range of273-320 mg/dL, upper end of normal 263 mg/dL) that returned to normal by15 days postinjection. Similarly, chloride levels were slightlyelevated, ranging from 116 to 127 mmol/L (upper end of normal range 115mmol/L) in the 2 highest dosage groups (57% in the 2×8 mg/kg group and100% of the mice in the 2×12 mg/kg group), and remained elevated out to15 days postinjection. This also could be indicative of gastrointestinaltoxicity, because most chloride is obtained through absorption by thegut; however, at termination, there was no histologic evidence of tissuedamage in any organ system examined (not shown).

Because mice do not express Trop-2 bound by hRS7, a more suitable modelwas required to determine the potential of the hRS7 conjugate forclinical use. Immunohistology studies revealed binding in multipletissues in both humans and Cynomolgus monkeys (breast, eye,gastrointestinal tract, kidney, lung, ovary, fallopian tube, pancreas,parathyroid, prostate, salivary gland, skin, thymus, thyroid, tonsil,ureter, urinary bladder, and uterus; not shown). Based on thiscross-reactivity, a tolerability study was performed in monkeys.

The group receiving 2×0.96 mg SN-38/kg of hRS7-CL2A-SN-38 had nosignificant clinical events following the infusion and through thetermination of the study. Weight loss did not exceed 7.3% and returnedto acclimation weights by day 15. Transient decreases were noted in mostof the blood count data (not shown), but values did not fall belownormal ranges. No abnormal values were found in the serum chemistries.Histopathology of the animals necropsied on day 11 (8 days after lastinjection) showed microscopic changes in hematopoietic organs (thymus,mandibular and mesenteric lymph nodes, spleen, and bone marrow),gastrointestinal organs (stomach, duodenum, jejunum, ileum, cecum,colon, and rectum), female reproductive organs (ovary, uterus, andvagina), and at the injection site. These changes ranged from minimal tomoderate and were fully reversed at the end of the recovery period (day32) in all tissues, except in the thymus and gastrointestinal tract,which were trending towards full recovery at this later timepoint.

At the 2×1.92 mg SN-38/kg dose level of the conjugate, there was 1 deatharising from gastrointestinal complications and bone marrow suppression,and other animals within this group showed similar, but more severeadverse events than the 2×0.96 mg/kg group. These data indicate thatdose-limiting toxicities were identical to that of irinotecan; namely,intestinal and hematologic. Thus, the MTD for hRS7-CL2A-SN-38 liesbetween 2×0.96 and 1.92 mg SN-38/kg, which represents a human equivalentdose of 2×0.3 to 0.6 mg/kg SN-38.

Discussion

Trop-2 is a protein expressed on many epithelial tumors, including lung,breast, colorectal, pancreas, prostate, and ovarian cancers, making it apotentially important target for delivering cytotoxic agents. The RS7antibody internalizes when bound to Trop-2 (Shih et al., 1995, CancerRes 55:5857s-63s), which enables direct intracellular delivery ofcytotoxics.

Conjugation of chemotherapeutic drugs to antibodies has been exploredfor over 30 years. Because a substantial portion of an ADC is notprocessed by the tumor, but by normal tissues, there is a risk thatthese agents will be too toxic to normal organ systems before reachingthe therapeutic level in tumors. As with any therapeutic, thetherapeutic window is a key factor determining the potential of an ADC,and thus rather than examining “ultratoxic” drugs, we chose SN-38 as thedrug component of the Trop-2-targeted ADC.

SN-38 is a potent topoisomerase-I inhibitor, with IC₅₀ values in thenanomolar range in several cell lines. It is the active form of theprodrug, irinotecan, that is used for the treatment of colorectalcancer, and which also has activity in lung, breast, and brain cancers.We reasoned that a directly targeted SN-38, in the form of an ADC, wouldbe a significantly improved therapeutic over CPT-11, by overcoming thelatter's low and patient-variable bioconversion to active SN-38.

The Phe-Lys peptide inserted in the original CL2 derivative allowed forpossible cleavage via cathepsin B. In an effort to simplify thesynthetic process, in CL2A, phenylalanine was eliminated, and thus thecathepsin B cleavage site was removed. Interestingly, this product had abetter-defined chromatographic profile compared to the broad profileobtained with CL2 (not shown), but more importantly, this change had nonegative impact on the conjugate's binding, stability, or potency inside-by-side testing. These data suggest that SN-38 in CL2 was releasedfrom the conjugate primarily by the cleavage at the pH-sensitive benzylcarbonate bond to SN-38's lactone ring and not the cathepsin B cleavagesite.

In vitro cytotoxicity of hRS7 ADC against a range of solid tumor celllines consistently had IC₅₀ values in the nmol/L range. However, cellsexposed to free SN-38 demonstrated a lower IC₅₀ value compared to theADC. This disparity between free and conjugated SN-38 was also reportedfor ENZ-2208 (Sapra et al., 2008, Clin Cancer Res 14:1888-96) and NK012(Koizumi et al., 2006, Cancer Res 66:10048-56). ENZ-2208 utilizes abranched PEG to link about 3.5 to 4 molecules of SN-38 per PEG, whereasNK012 is a micelle nanoparticle containing 20% SN-38 by weight. With ourADC, this disparity (i.e., ratio of potency with free vs. conjugatedSN-38) decreased as the Trop-2 expression levels increased in the tumorcells, suggesting an advantage to targeted delivery of the drug. Interms of in vitro serum stability, both the CL2- and CL2A-SN-38 forms ofhRS7-SN-38 yielded a t/_(1/2) of ˜20 hours, which is in contrast to theshort t/_(1/2) of 12.3 minutes reported for ENZ-2208 (Zhao et al., 2008,Bioconjug Chem 19:849-59), but similar to the 57% release of SN-38 fromNK012 under physiological conditions after 24 hours (Koizumi et al.,2006, Cancer Res 66:10048-56).

Treatment of tumor-bearing mice with hRS7-SN-38 (either with CL2-SN-38or CL2A-SN-38) significantly inhibited tumor growth in 5 different tumormodels. In 4 of them, tumor regressions were observed, and in the caseof Calu-3, all mice receiving the highest dose of hRS7-SN-38 weretumor-free at the conclusion of study. Unlike in humans, irinotecan isvery efficiently converted to SN-38 by a plasma esterase in mice, with agreater than 50% conversion rate, and yielding higher efficacy in micethan in humans. When irinotecan was administered at 10-fold higher orequivalent SN-38 levels, hRS7-SN-38 was significantly better incontrolling tumor growth. Only when irinotecan was administered at itsMTD of 24 mg/kg q2dx5 (37.5-fold more SN-38) did it equal theeffectiveness of hRS7-SN-38. In patients, we would expect this advantageto favor hRS7-CL2A-SN-38 even more, because the bioconversion ofirinotecan would be substantially lower.

We also showed in some antigen-expressing cell lines, such as SK-MES-1,that using an antigen-binding ADC does not guarantee better therapeuticresponses than a nonbinding, irrelevant conjugate. This is not anunusual or unexpected finding. Indeed, the nonbinding SN-38 conjugatesmentioned earlier enhance therapeutic activity when compared toirinotecan, and so an irrelevant IgG-SN-38 conjugate is expected to havesome activity. This is related to the fact that tumors have immature,leaky vessels that allow the passage of macromolecules better thannormal tissues. With our conjugate, 50% of the SN-38 will be released in˜13 hours when the pH is lowered to a level mimicking lysosomal levels(e.g., pH 5.3 at 37° C.; data not shown), whereas at the neutral pH ofserum, the release rate is reduced nearly 2-fold. If an irrelevantconjugate enters an acidic tumor microenvironment, it is expected torelease some SN-38 locally. Other factors, such as tumor physiology andinnate sensitivities to the drug, will also play a role in defining this“baseline” activity. However, a specific conjugate with a longerresidence time should have enhanced potency over this baseline responseas long as there is ample antigen to capture the specific antibody.Biodistribution studies in the SK-MES-1 model also showed that if tumorantigen becomes saturated as a consequence of successive dosing, tumoruptake of the specific conjugate is reduced, which yields therapeuticresults similar to that found with an irrelevant conjugate.

Although it is challenging to make direct comparisons between our ADCand the published reports of other SN-38 delivery agents, some generalobservations can be made. In our therapy studies, the highest individualdose was 0.4 mg/kg of SN-38. In the Calu-3 model, only 4 injections weregiven for a total cumulative dose of 1.6 mg/kg SN-38 or 32 μg SN-38 in a20 g mouse. Multiple studies with ENZ-2208 were done using its MTD of 10mg/kg×5, and preclinical studies with NK012 involved its MTD of 30mg/kg×3. Thus, significant antitumor effects were obtained withhRS7-SN-38 at 30-fold and 55-fold less SN-38 equivalents than thereported doses in ENZ-2208 and NK012, respectively. Even with 10-foldless hRS7 ADC (0.04 mg/kg), significant antitumor effects were observed,whereas lower doses of ENZ-2208 were not presented, and when the NK012dose was lowered 4-fold to 7.5 mg/kg, efficacy was lost (Koizumi et al.,2006, Cancer Res 66:10048-56). Normal mice showed no acute toxicity witha cumulative dose over 1 week of 24 mg/kg SN-38 (1,500 mg/kg of theconjugate), indicating that the MTD was higher. Thus, tumor-bearinganimals were effectively treated with 7.5- to 15-fold lower amounts ofSN-38 equivalents.

As a topoisomerase-I inhibitor, SN-38 induces significant damage to acell's DNA, with upregulation of p53 and p21^(WAF1/CiP1) resulting incaspase activation and cleavage of PARP. When we exposed BxPC-3 andCalu-3 cells to our ADC, both p53 and p21^(WAF1/Cip1) were upregulatedabove basal levels. In addition, PARP cleavage was also evident in bothcell lines, confirming an apoptotic event in these cells. Of interestwas the higher upregulation of p21^(WAF1/Cip1) in BxPC-3 and Calu-3relative to p53 by both free SN-38 and our hRS7-SN-38. This may beindicative of the mutational status of p53 in these 2 cell lines and theuse of a p53-independent pathway for p21^(WAF1/Cip1)-mediated apoptosis.

An interesting observation was the early upregulation of p53 in bothBxPC-3 and Calu-3 at 24 hours mediated by the hRS7-ADC relative to freeSN-38. Even the naked hRS7 IgG could upregulate p53 in these cell lines,although only after a 48-hour exposure. Trop-2 overexpression andcross-linking by antibodies has been linked to several MAPK-relatedsignaling events, as well as intracellular calcium release. Whilebinding of hRS7 was not sufficient to induce apoptosis in BxPC-3 andCalu-3, as evidenced by the lack of PARP cleavage, it may be enough toprime a cell, such that the inclusion of SN-38 conjugated to hRS7 maylead to a greater effect on tumor growth inhibition. Studies arecurrently underway to understand which pathways are involved withhRS7-delivery of SN-38 and how they may differ from free SN-38, and whateffect p53 status may play in this signaling.

Biodistribution studies revealed the hRS7-CL2A-SN-38 had similar tumoruptake as the parental hRS7 IgG, but cleared substantially faster with2-fold higher hepatic uptake, which may be due to the hydrophobicity ofSN-38. With the ADC being cleared through the liver, hepatic andgastrointestinal toxicities were expected to be dose limiting. Althoughmice had evidence of increased hepatic transaminases, gastrointestinaltoxicity was mild at best, with only transient loss in weight and noabnormalities noted upon histopathologic examination. Interestingly, nohematological toxicity was noted. However, monkeys showed an identicaltoxicity profile as expected for irinotecan, with gastrointestinal andhematological toxicity being dose-limiting.

Because Trop-2 recognized by hRS7 is not expressed in mice, it wascritically important to perform toxicity studies in monkeys that have asimilar tissue expression of Trop-2 as humans. Monkeys tolerated 0.96mg/kg/dose (˜12 mg/m²) with mild and reversible toxicity, whichextrapolates to a human dose of ˜0.3 mg/kg/dose (˜11 mg/m²). In a PhaseI clinical trial of NK012, patients with solid tumors tolerated 28 mg/m²of SN-38 every 3 weeks with Grade 4 neutropenia as dose-limitingtoxicity (Hamaguchi et al., 2010, Clin Cancer Res 16:5058-66).Similarly, Phase I clinical trials with ENZ-2208 revealed dose-limitingfebrile neutropenia, with a recommendation to administer 10 mg/m² every3 weeks or 16 mg/m² if patients were administered G-CSF. Because monkeystolerated a cumulative human equivalent dose of 22 mg/m², it is possiblethat even though hRS7 binds to a number of normal tissues, the MTD for asingle treatment of the hRS7 ADC could be similar to that of the othernontargeting SN-38 agents. Indeed, the specificity of the anti-Trop-2antibody did not appear to play a role in defining the DLT, because thetoxicity profile was similar to that of irinotecan. More importantly, ifantitumor activity can be achieved in humans as in mice that respondedwith human equivalent dose of just at 0.03 mg SN-38 equivalents/kg/dose,then significant antitumor responses could be realized clinically.

In conclusion, toxicology studies in monkeys, combined with in vivohuman cancer xenograft models in mice, have indicated that this ADCtargeting Trop-2 is an effective therapeutic in several tumors ofdifferent epithelial origin.

Example 10 Efficacy of Anti-Trop-2 Antibody Conjugated to a Prodrug Formof 2-Pyrrolinodoxorubicin (2-PDox)

A prodrug form of 2-PDox (referred to as pro-2-PDox) was prepared andconjugated to antibodies as disclosed in U.S. patent application Ser.No. 14/175,089 (Example 1 of which is incorporated herein by reference).Unless otherwise stated below, the number of drug moieties per antibodymolecule was in the range of about 6.5 to about 7.5.

In vitro cell-binding studies—Retention of antibody binding wasconfirmed by cell binding assays comparing binding of the conjugated tothe unconjugated antibody (Chari, 2008, Acc Chem Res 41:98-107). Thepotency of the conjugate was tested in a 4-day MTS assay usingappropriate target cells. The anti-Trop-2 ADC (hRS7-pro-2-PDox)exhibited IC₅₀ values of 0.35-1.09 nM in gastric (NCI-N87), pancreatic(Capan-1), and breast (MDA-MB-468) human cancer cell lines, with freedrug exhibiting 0.02-0.07 nM potency in the same cell lines. Inadditional studies, hRS7-pro-2-PDox was observed to be cytotoxic toMDA-MB-468, AGS, NCI-N87 and Capan-1 solid tumor cell lines (not shown).

No significant difference in binding of the antibody moiety to NCI-N87gastric carcinoma cells was observed between unconjugated hRS7 andpro-2-PDox-hRS7 conjugated to 6 molecules of pro-2-PDox per antibody(not shown). It is concluded that conjugation of pro-2-PDox toantibodies does not affect antibody-antigen binding activity.

Serum stability—Serum stability of anti-Trop-2 ADC (hRS7-pro-2-PDox) wasdetermined by incubation in human serum at a concentration of 0.2 mg/mLat 37° C. The incubate was analyzed by HPLC using butyl hydrophobicinteraction chromatography (HIC). The analysis showed that there was norelease of free drug from the conjugate, suggesting high serum stabilityof the conjugate. When the same experiment was repeated withhRS7-doxorubicin conjugate, containing the same cleavable linker ashRS7-pro-2-PDox, and where the free drug was independently verified tobe released with a half-life of 96 h, clear formation of a peakcorresponding to free doxorubicin was seen on HIC HPLC.

Surprisingly, it was determined that the pro-2-PDox conjugate was heldtightly to the antibody because it cross-linked the peptide chains ofthe antibody together. The cross-linking stabilizes the attachment ofthe drug to the antibody so that the drug is only releasedintracellularly after the antibody is metabolized. The cross-linkingassists in minimizing toxicity, for example cardiotoxicity, that wouldresult from release of free drug in circulation. Previous use of 2-PDoxpeptide conjugates failed because the drug cross-linked the peptide toother proteins or peptides in vivo. With the present anti-Trop-2 ADC,the pro-2-PDox is attached to interchain disulfide thiol groups while inthe prodrug form. The prodrug protection is rapidly removed in vivo soonafter injection and the resulting 2-PDox portion of the conjugatecross-links the peptide chains of the antibody, forming intramolecularcross-linking within the antibody molecule. This both stabilizes the ADCand prevents cross-linking to other molecules in circulation.

In vivo preclinical studies—Tumor size was determined by calipermeasurements of length (L) and width (W) with tumor volume calculated as(L×W²)/2. Tumors were measured and mice weighed twice a week. Mice wereeuthanized if their tumors reached >1 cm³ in size, lost greater than 15%of their starting body weight, or otherwise became moribund. Statisticalanalysis for the tumor growth data was based on area under the curve(AUC) and survival time. Profiles of individual tumor growth wereobtained through linear curve modeling. An f-test was employed todetermine equality of variance between groups prior to statisticalanalysis of growth curves. A two-tailed t-test was used to assessstatistical significance between all the various treatment groups andnon-specific controls. For the saline control analysis a one-tailedt-test was used to assess significance. Survival studies were analyzedusing Kaplan-Meier plots (log-rank analysis), using the Prism GraphPadSoftware (v4.03) software package (Advanced Graphics Software, Inc.;Encinitas, Calif.). All doses in preclinical experiments are expressedin antibody amounts. In terms of drug, 100 μg of antibody (5 mg/kg) in a20-g mouse, for example, carries 1.4 μg-2.8 μg (0.14-0.17 mg/kg) ofpro-2-PDox equivalent dose when using an ADC with 3-6 drugs/IgG.

A single i.v. dose of ≥300 μg [˜10 μg of pro-2-PDox] of the anti-Trop-2ADC was lethal, but 4 doses of 45 μg given in 2 weeks were tolerated byall animals. Using this dosing regimen, we examined the therapeuticeffect of anti-Trop-2 hRS7-pro-2-PDox in 2 human tumor xenograft models,Capan-1 (pancreatic cancer) and NCI-N87 (gastric cancer). Therapy began7 days after tumor transplantation in nude mice. In the established,7-day-old, Capan-1 model, 100% of established tumors quickly regressed,with no evidence of re-growth (not shown). This result was reproduced ina repeat experiment (not shown). The anti-Trop-2 conjugate of pro-2-PDoxwas much more effective than the same drug conjugated to an antibody(hMN-14) against CEACAM5, which is also expressed in pancreatic cancer,or an antibody against CD20 (hA20), which is not. All treatments weresuperior to the saline control.

Similar results were observed in the established NCI-N87 model (notshown), where a 2^(nd) course of therapy, administered after day 70, wassafely tolerated and led to further regressions of residual tumor (notshown). The internalizing hRS7-SN-38 conjugate, targeting Trop-2,provided better therapeutic responses than a conjugate of a poorlyinternalizing anti-CEACAM5 antibody, hMN-14 (not shown). A non-targetedanti-CD20 ADC, hA20-pro-2-PDox, was ineffective, indicating selectivetherapeutic efficacy (not shown). Data from a breast cancer xenograft(MDA-MB-468) and a second pancreatic cancer xenograft (not shown) showedthe same pattern, with the anti-Trop-2 ADC significantly moreefficacious compared to non-targeting ADC or saline control. In bothcases, administration of anti-Trop-2 ADC produced a clear inhibition oftumor growth to the end of the study.

PK and toxicity of hRS7-pro-2-PDox with substitutions of 6.8 or 3.7drug/IgG—Antibody-drug conjugates (ADCs) carrying as much as 8ultratoxic drugs/MAb are known to clear faster than unmodified MAb andto increase off-target toxicity, a finding that has led to the currenttrends to use drug substitutions of ≤4 (Hamblett et al., 2004, ClinCancer Res 10:7063-70). ADCs were prepared and evaluated with meandrug/MAb substitution ratios (MSRs) of ˜6:1 and ˜3:1. Groups of normalmice (n=5) were administered, i.v., single doses of unmodified hRS7 orhRS7-pro-2-PDox with drug substitution of 6.8 or 3.7 (same proteindose), and serum samples were collected at 30 min, 4 h, 24 h, 72 h, and168 h post-injection. These were analyzed by ELISA for antibodyconcentration. There were no significant differences in serumconcentrations at various times, indicating that these showed similarclearance from the blood. The PK parameters (Cmax, AUC, etc.) were alsosimilar. ADCs with either higher or lower drug substitution had similartolerability in nude mice, when the administered at the same dose ofconjugated drug.

Therapeutic Efficacy at Minimum Effective Dose (MED)—Anti-Trop-2 ADC(hRS7-pro-2-PDox), was evaluated in nude mice bearing NCI-N87 humangastric cancer xenografts by administering a single bolus protein doseof 9 mg/kg, 6.75 mg/kg, 4.5 mg/kg, 2.25 mg/kg, or 1 mg/kg. The therapywas started when the mean tumor volume (mTV) was 0.256 cm³. On day 21,mTV in the saline control group (non-treatment group) was 0.801±0.181cm³ which was significantly larger than that in mice treated with 9,6.75, 4.5, or 2.25 mg/kg dose with mTV of 0.211±0.042 cm³, 0.239±0.0.054cm³, 0.264±0.087 cm³, and 0.567±0.179 cm³, respectively (P<0.0047, onetailed t-test). From these, the minimum effective dose was estimated tobe 2.25 mg/kg, while 9 mg/kg represented MTD.

Example 11 Anti-Trop-2 ADC Comprising hRS7 and Paclitaxel

A new antibody-drug conjugate (ADC) was made by conjugating paclitaxel(TAXOL®) to the hRS7 anti-human Trop-2 antibody (hRS7-paclitaxel). Thefinal product had a mean drug to antibody substitution ratio of 2.2.This ADC was tested in vitro using two different Trop-2-positive celllines as targets: BxPC-3 (human pancreatic adenocarcinoma) andMDA-MB-468 (human triple negative breast carcinoma). One day prior toadding the ADC, cells were harvested from tissue culture and plated into96-well plates at 2000 cells per well. The next day cells were exposedto free paclitaxel (6.1×10⁻¹¹ to 4×10⁻⁶ M) or the drug-equivalent ofhRS7-paclitaxel. For comparison, hRS7-SN-38 and free SN-38 were alsotested at a range of 3.84×10⁻¹² to 2.5×10⁻⁷ M. Plates were incubated at37° C. for 96 h. After this incubation period, an MTS substrate wasadded to all of the plates and read for color development at half-hourintervals until untreated control wells had an OD_(492nm) reading ofapproximately 1.0. Growth inhibition was measured as a percent of growthrelative to untreated cells using Microsoft Excel and Prism software(non-linear regression to generate sigmoidal dose response curves whichyield IC₅₀-values).

The hRS7-paclitaxel ADC exhibited cytotoxic activity in the MDA-MB-468breast cell line (not shown), with an IC₅₀-value approximately 4.5-foldhigher than hRS7-SN-38. The free paclitaxel was much more potent thanthe free SN-38 (not shown). While the IC₅₀ for free SN-38 was 1.54×10⁻⁹M, the IC₅₀ for free paclitaxel was less than 6.1×10⁻¹¹ M. Similarresults were obtained for the BxPC-3 pancreatic cell line (not shown) inwhich the hRS7-paclitaxel ADC had an IC₅₀-value approximately 2.8-foldhigher than the hRS7-SN-38 ADC. These results show the efficacy ofanti-Trop-2 conjugated paclitaxel in vitro, with IC₅₀-values in thenanomolar range, similar to the hRS7-SN-38 ADC.

Example 12 Cytotoxicity of Anti-Trop-2 ADC (MAB650-SN-38)

A novel anti-Trop-2 ADC was made with SN-38 and MAB650, yielding a meandrug to antibody substitution ratio of 6.89. Cytotoxicity assays wereperformed to compare the MAB650-SN-38 and hRS7-SN-38 ADCs using twodifferent human pancreatic adenocarcinoma cell lines (BxPC-3 andCapan-1) and a human triple negative breast carcinoma cell line(MDA-MB-468) as targets.

One day prior to adding the ADCs, cells were harvested from tissueculture and plated into 96-well plates. The next day cells were exposedto hRS7-SN-38, MAB650-SN-38, and free SN-38 at a drug range of3.84×10⁻¹² to 2.5×10⁻⁷ M. Unconjugated MAB650 was used as a control atprotein equivalent doses as the MAB650-SN-38. Plates were incubated at37° C. for 96 h. After this incubation period, an MTS substrate wasadded to all of the plates and read for color development at half-hourintervals until an OD_(492nm) of approximately 1.0 was reached for theuntreated cells. Growth inhibition was measured as a percent of growthrelative to untreated cells using Microsoft Excel and Prism software(non-linear regression to generate sigmoidal dose response curves whichyield IC₅₀-values.

hRS7-SN-38 and MAB650-SN-38 had similar growth-inhibitory effects (notshown) with IC₅₀-values in the low nM range which is typical forSN-38-ADCs in these cell lines. In the human Capan-1 pancreaticadenocarcinoma cell line (not shown), the hRS7-SN-38 ADC showed an IC₅₀of 3.5 nM, compared to 4.1 nM for the MAB650-SN-38 ADC and 1.0 nM forfree SN-38. In the human BxPC-3 pancreatic adenocarcinoma cell line (notshown), the hRS7-SN-38 ADC showed an IC₅₀ of 2.6 nM, compared to 3.0 nMfor the MAB650-SN-38 ADC and 1.0 nM for free SN-38. In the human NCI-N87gastric adenocarcinoma cell line (not shown), the hRS7-SN-38 ADC showedan IC₅₀ of 3.6 nM, compared to 4.1 nM for the MAB650-SN-38 ADC and 4.3nM for free SN-38.

In summary, in these in vitro assays, the SN-38 conjugates of twoanti-Trop-2 antibodies, hRS7 and MAB650, showed equal efficacies againstseveral tumor cell lines, which was similar to that of free SN-38.Because the targeting function of the anti-Trop-2 antibodies would be amuch more significant factor in vivo than in vitro, the data supportthat anti-Trop-2-SN-38 ADCs as a class would be highly efficacious invivo, as demonstrated in the Examples above for hRS7-SN-38.

Example 13 Cytotoxicity of Anti-Trop-2 ADC (162-46.2-SN-38)

A novel anti-Trop-2 ADC was made with SN-38 and 162-46.2, yielding adrug to antibody substitution ratio of 6.14. Cytotoxicity assays wereperformed to compare the 162-46.2-SN-38 and hRS7-SN-38 ADCs using twodifferent Trop-2-positive cell lines as targets, the BxPC-3 humanpancreatic adenocarcinoma and the MDA-MB-468 human triple negativebreast carcinoma.

One day prior to adding the ADC, cells were harvested from tissueculture and plated into 96-well plates at 2000 cells per well. The nextday cells were exposed to hRS7-SN-38, 162-46.2-SN-38, or free SN-38 at adrug range of 3.84×10⁻¹² to 2.5×10⁻⁷M. Unconjugated 162-46.2 and hRS7were used as controls at the same protein equivalent doses as the162-46.2-SN-38 and hRS7-SN-38, respectively. Plates were incubated at37° C. for 96 h. After this incubation period, an MTS substrate wasadded to all of the plates and read for color development at half-hourintervals until untreated control wells had an OD492 reading ofapproximately 1.0. Growth inhibition was measured as a percent of growthrelative to untreated cells using Microsoft Excel and Prism software(non-linear regression to generate sigmoidal dose response curves whichyield IC₅₀-values).

The 162-46.2-SN-38 ADC had a similar IC₅₀-values when compared tohRS7-SN-38 (not shown). When tested against the BxPC-3 human pancreaticadenocarcinoma cell line (not shown), hRS7-SN-38 had an IC₅₀ of 5.8 nM,compared to 10.6 nM for 162-46.2-SN-38 and 1.6 nM for free SN-38. Whentested against the MDA-MB-468 human breast adenocarcinoma cell line (notshown), hRS7-SN-38 had an IC₅₀ of 3.9 nM, compared to 6.1 nM for162-46.2-SN-38 and 0.8 nM for free SN-38. The free antibodies aloneshowed little cytotoxicity to either Trop-2 positive cancer cell line.

In summary, comparing the efficacies in vitro of three differentanti-Trop-2 antibodies conjugated to the same cytotoxic drug, all threeADCs exhibited equivalent cytotoxic effects against a variety of Trop-2positive cancer cell lines. These data support that the class ofanti-Trop-2 antibodies, incorporated into drug-conjugated ADCs, areeffective anti-cancer therapeutic agents for Trop-2 expressing solidtumors.

Example 14 Clinical Trials With IMMU-132 Anti-Trop-2 ADC Comprising hRS7Antibody Conjugated to SN-38

Summary

The present Example reports results from a phase I clinical trial andongoing phase II extension with IMMU-132, an ADC of the internalizing,humanized, hRS7 anti-Trop-2 antibody conjugated by a pH-sensitive linkerto SN-38 (mean drug-antibody ratio=7.6). Trop-2 is a type Itransmembrane, calcium-transducing, protein expressed at high density(˜1×10⁵), frequency, and specificity by many human carcinomas, withlimited normal tissue expression. Preclinical studies in nude micebearing Capan-1 human pancreatic tumor xenografts have revealed IMMU-132is capable of delivering as much as 120-fold more SN-38 to tumor thanderived from a maximally tolerated irinotecan therapy.

The present Example reports the initial Phase I trial of 25 patients(pts) who had failed multiple prior therapies (some includingtopoisomerase-I/II inhibiting drugs), and the ongoing Phase II extensionnow reporting on 69 pts, including in colorectal (CRC), small-cell andnon-small cell lung (SCLC, NSCLC, respectively), triple-negative breast(TNBC), pancreatic (PDC), esophageal, and other cancers.

As discussed in detail below, Trop-2 was not detected in serum, but wasstrongly expressed (≥2⁺) in most archived tumors. In a 3+3 trial design,IMMU-132 was given on days 1 and 8 in repeated 21-day cycles, startingat 8 mg/kg/dose, then 12 and 18 mg/kg before dose-limiting neutropenia.To optimize cumulative treatment with minimal delays, phase II isfocusing on 8 and 10 mg/kg (n=30 and 14, respectively). In 49 ptsreporting related AE at this time, neutropenia ≥G3 occurred in 28% (4%G4). Most common non-hematological toxicities initially in these ptshave been fatigue (55%; ≥G3=9%), nausea (53%; ≥G3=0%), diarrhea (47%;≥G3=9%), alopecia (40%), and vomiting (32%; ≥G3=2%). Homozygous UGT1A1*28/*28 was found in 6 pts, 2 of whom had more severe hematological andGI toxicities. In the Phase I and the expansion phases, there are now 48pts (excluding PDC) who are assessable by RECIST/CT for best response.Seven (15%) of the patients had a partial response (PR), includingpatients with CRC (N=1), TNBC (N=2), SCLC (N=2), NSCLC (N=1), andesophageal cancers (N=1), and another 27 pts (56%) had stable disease(SD), for a total of 38 pts (79%) with disease response; 8 of 13CT-assessable PDC pts (62%) had SD, with a median time to progression(TTP) of 12.7 wks compared to 8.0 weeks in their last prior therapy. TheTTP for the remaining 48 pts is 12.6+ wks (range 6.0 to 51.4 wks).Plasma CEA and CA19-9 correlated with responses. No anti-hRS7 oranti-SN-38 antibodies were detected despite dosing over months. Theconjugate cleared from the serum within 3 days, consistent with in vivoanimal studies where 50% of the SN-38 was released daily, with >95% ofthe SN-38 in the serum being bound to the IgG in a non-glucoronidatedform, and at concentrations as much as 100-fold higher than SN-38reported in patients given irinotecan. These results show that thehRS7-SN-38-containing ADC is therapeutically active in metastatic solidcancers, with manageable diarrhea and neutropenia.

Pharmacokinetics

Two ELISA methods were used to measure the clearance of the IgG (capturewith anti-hRS7 idiotype antibody) and the intact conjugate (capture withanti-SN-38 IgG/probe with anti-hRS7 idiotype antibody). SN-38 wasmeasured by HPLC. Total IMMU-132 fraction (intact conjugate) clearedmore quickly than the IgG (not shown), reflecting known gradual releaseof SN-38 from the conjugate. HPLC determination of SN-38 (Unbound andTOTAL) showed >95% the SN-38 in the serum was bound to the IgG. Lowconcentrations of SN-38G suggest SN-38 bound to the IgG is protectedfrom glucoronidation. Comparison of ELISA for conjugate and SN-38 HPLCrevealed both overlap, suggesting the ELISA is a surrogate formonitoring SN-38 clearance.

A summary of the dosing regiment and patient poll is provided in Table12.

TABLE 12 Clinical Trial Parameters Dosing Once weekly for 2 weeksadministered every 21 days for regimen up to 8 cycles. In the initialenrollment, the planned dose was delayed and reduced if ≥G2treatment-related toxicity; protocol was amended to dose delay andreduction only in the event of ≥G3 toxicity. Dose level 8, 12, 18 mg/kg;later reduced to an intermediate dose cohorts level of 10 mg/kg. Cohortsize Standard Phase I [3 + 3] design; expansion includes 15 patients inselect cancers. DLT G4 ANC ≥7 d; ≥G3 febrile neutropenia of anyduration; G4 Plt ≥5 d; G4 Hgb; Grade 4 N/V/D any duration/G3 N/V/Dfor >48 h; G3 infusion-related reactions; related ≥G3 non-hematologicaltoxicity. Maximum Maximum dose where ≥2/6 patients tolerate 1^(st) 21-dAcceptable cycle w/o delay or reduction or ≥G3 toxicity. Dose (MAD)Patients Metastatic colorectal, pancreas, gastric, esophageal, lung(NSCLC, SCLC), triple-negative breast (TNBC), prostate, ovarian, renal,urinary bladder, head/neck, hepatocellular. Refractory/relapsed afterstandard treatment regimens for metastatic cancer. Prioririnotecan-containing therapy NOT required for enrollment. No bulkylesion >5 cm. Must be 4 weeks beyond any major surgery, and 2 weeksbeyond radiation or chemotherapy regimen. Gilbert's disease or known CNSmetastatic disease are excluded.

Clinical Trial Status

A total of 69 patients (including 25 patients in Phase I) with diversemetastatic cancers having a median of 3 prior therapies were reported.Eight patients had clinical progression and withdrew before CTassessment. Thirteen CT-assessable pancreatic cancer patients wereseparately reported. The median TTP (time to progression) in PDCpatients was 11.9 wks (range 2 to 21.4 wks) compared to median 8 wks TTPfor the preceding last therapy.

A total of 48 patients with diverse cancers had at least 1 CT-assessmentfrom which Best Response (not shown) and Time to Progression (TTP; notshown) were determined. To summarize the Best Response data, of 8assessable patients with TNBC (triple-negative breast cancer), therewere 2 PR (partial response), 4 SD (stable disease) and 2 PD(progressive disease) for a total response [PR+SD] of 6/8 (75%). ForSCLC (small cell lung cancer), of 4 assessable patients there were 2 PR,0 SD and 2 PD for a total response of 2/4 (50%). For CRC (colorectalcancer), of 18 assessable patients there were 1 PR, 11 SD and 6 PD for atotal response of 12/18 (67%). For esophageal cancer, of 4 assessablepatients there were 1 PR, 2 SD and 1 PD for a total response of ¾ (75%).For NSCLC (non-small cell lung cancer), of 5 assessable patients therewere 1 PR, 3 SD and 1 PD for a total response of ⅘ (80%). Over allpatients treated, of 48 assessable patients there were 7 PR, 27 SD and14 PD for a total response of 34/48 (71%). These results demonstratethat the anti-TROP-2 ADC (hRS7-SN-38) showed significant clinicalefficacy against a wide range of solid tumors in human patients.

The reported side effects of therapy (adverse events) are summarized inTable 13. As apparent from the data of Table 13, the therapeuticefficacy of hRS7-SN-38 was achieved at dosages of ADC showing anacceptably low level of adverse side effects.

TABLE 13 Related Adverse Events Listing for IMMU-132-01 Criteria: Total≥10% or ≥Grade 3 N = 47 patients TOTAL Grade 3 Grade 4 Fatigue 55% 4(9%) 0 Nausea 53% 0 0 Diarrhea 47% 4 (9%) 0 Neutropenia 43% 11 (24%) 2(4%) Alopecia 40% — — Vomiting 32% 1 (2%) 0 Anemia 13% 2 (4%) 0Dysgeusia 15% 0 0 Pyrexia 13% 0 0 Abdominal pain 11% 0 0 Hypokalemia 11%1 (2%) 0 WBC Decrease  6% 1 (2%) 0 Febrile Neutropenia  6% 1 (2%) 2 (4%)Deep vein thrombosis  2% 1 (2%) 0 Grading by CTCAE v 4.0

Exemplary partial responses to the anti-Trop-2 ADC were confirmed by CTdata (not shown). As an exemplary PR in CRC, a 62 year-old woman firstdiagnosed with CRC underwent a primary hemicolectomy. Four months later,she had a hepatic resection for liver metastases and received 7 mos oftreatment with FOLFOX and 1 mo SFU. She presented with multiple lesionsprimarily in the liver (3+ Trop-2 by immunohistology), entering thehRS7-SN-38 trial at a starting dose of 8 mg/kg about 1 year afterinitial diagnosis. On her first CT assessment, a PR was achieved, with a37% reduction in target lesions (not shown). The patient continuedtreatment, achieving a maximum reduction of 65% decrease after 10 monthsof treatment (not shown) with decrease in CEA from 781 ng/mL to 26.5ng/mL), before progressing 3 months later.

As an exemplary PR in NSCLC, a 65 year-old male was diagnosed with stageIIIB NSCLC (sq. cell). Initial treatment of caboplatin/etoposide (3 mo)in concert with 7000 cGy XRT resulted in a response lasting 10 mo. Hewas then started on Tarceva maintenance therapy, which he continueduntil he was considered for IMMU-132 trial, in addition to undergoing alumbar laminectomy. He received first dose of IMMU-132 after 5 months ofTarceva, presenting at the time with a 5.6 cm lesion in the right lungwith abundant pleural effusion. He had just completed his 6^(th) dosetwo months later when the first CT showed the primary target lesionreduced to 3.2 cm (not shown).

As an exemplary PR in SCLC, a 65 year-old woman was diagnosed withpoorly differentiated SCLC. After receiving carboplatin/etoposide(Topo-II inhibitor) that ended after 2 months with no response, followedwith topotecan (Topo-I inhibitor) that ended after 2 months, also withno response, she received local XRT (3000 cGy) that ended 1 month later.However, by the following month progression had continued. The patientstarted with IMMU-132 the next month (12 mg/kg; reduced to 6.8 mg/kg;Trop-2 expression 3+), and after two months of IMMU-132, a 38% reductionin target lesions, including a substantial reduction in the main lunglesion occurred (not shown). The patient progressed 3 months later afterreceiving 12 doses.

These results are significant in that they demonstrate that theanti-Trop-2 ADC was efficacious, even in patients who had failed orprogressed after multiple previous therapies.

In conclusion, at the dosages used, the primary toxicity was amanageable neutropenia, with few Grade 3 toxicities. IMMU-132 showedevidence of activity (PR and durable SD) in relapsed/refractory patientswith triple-negative breast cancer, small cell lung cancer, non-smallcell lung cancer, colorectal cancer and esophageal cancer, includingpatients with a previous history of relapsing on topoisomerase-Iinhibitor therapy. These results show efficacy of the anti-Trop-2 ADC ina wide range of cancers that are resistant to existing therapies.

Example 15 Anti-CD22 (Epratuzumab) Conjugated-SN-38 for the Therapy ofHematologic Malignancies

Abstract

We previously found that slowly internalizing antibodies conjugated withSN-38 could be used successfully when prepared with a linker that allowsapproximately 50% of the IgG-bound SN-38 to dissociate in serum every 24hours. In this study, the efficacy of SN-38 conjugates prepared withepratuzumab (rapidly internalizing) and veltuzumab (slowlyinternalizing), humanized anti-CD22 and anti-CD20 IgG, respectively, wasexamined for the treatment of B-cell malignancies. Both antibody-drugconjugates had similar nanomolar activity against a variety of humanlymphoma/leukemia cell lines, but slow release of SN-38 compromisedpotency discrimination in vitro even against an irrelevant conjugate.When SN-38 was stably linked to the anti-CD22 conjugate, its potency wasreduced 40- to 55-fold. Therefore, further studies were conducted onlywith the less stable, slowly dissociating linker. In vivo, similarantitumor activity was found between CD22 and CD20 antibody-drugconjugate in mice-bearing Ramos xenografts, even though Ramos expressed15-fold more CD20 than CD22, suggesting that the internalization of theepratuzumab-SN-38 conjugate (Emab-SN-38) enhanced its activity.Emab-SN-38 was more efficacious than a nonbinding, irrelevant IgG-SN-38conjugate in vivo, eliminating a majority of well-established Ramosxenografts at nontoxic doses. In vitro and in vivo studies showed thatEmab-SN-38 could be combined with unconjugated veltuzumab for a moreeffective treatment. Thus, Emab-SN-38 is active in lymphoma and leukemiaat doses well below toxic levels and therefore represents a newpromising agent with therapeutic potential alone or combined withanti-CD20 antibody therapy. (Sharkey et al., 2011, Mol Cancer Ther11:224-34.)

Introduction

A significant effort has focused on the biologic therapy of leukemia andlymphoma, where unconjugated antibodies (e.g., rituximab, alemtuzumab,ofatumumab), radioimmunoconjugates (⁹⁰Y-ibritumomab tiuxetan,¹³¹I-tositumomab), and a drug conjugate (gemtuzumab ozogamicin) receivedU.S. Food and Drug Administration (FDA) approval. Another antibody-drugconjugate (ADC), brentuximab vedotin (SGN-35; anti-CD30-auristatin E),recently received accelerated approval by the FDA for Hodgkin lymphomaand anaplastic large-cell lymphomas. There are also a number of otherADCs in preclinical and clinical development that target CD19, CD22,CD37, CD74, and CD79b.

Antibodies against all of these targets are logical choices for carriersof drugs, because they are internalizing. Internalization andspecificity of CD22 have made it a particularly important target forleukemia and lymphomas, with at least 3 different anti-CD22 conjugatesin clinical investigation, including CMC-544 (acid-labile-conjugatedcalicheamicin), an anti-CD22-maytansine conjugate (stably linkedMCC-DM1), and CAT-3888 (formally BL22; a Pseudomonas exotoxinsingle-chain fusion protein). The active agent in all of theseconjugates has subnanomolar potency (i.e., so called ultra-toxics).

We recently developed methods to conjugate antibodies with SN-38, atopoisomerase I inhibitor with low nanomolar potency that is derivedfrom the prodrug, irinotecan (Govindan et al., 2009, Clin Cancer Res15:6052-62; Moon et al., 2008, J Med Chem 51:6916-26). Four SN-38linkage chemistries were examined initially using conjugates preparedwith a slowly internalizing anti-CEACAM5 antibody (Govindan et al.,2009, Clin Cancer Res 15:6052-62; Moon et al., 2008, J Med Chem51:6916-26). The conjugates retained CEACAM5 binding but differed in thedissociation rate of SN-38 in human serum, with half-lives varying fromapproximately 10 to 67 hours (Govindan et al., 2009, Clin Cancer Res15:6052-62). Ultimately, the linker designated CL2, with intermediatestability (˜50% dissociated in 24-35 hours), was selected for furtherdevelopment. CL2 was modified recently, eliminating the phenylalanine inthe cathepsin B-cleavable dipeptide to simplify and improvemanufacturing yields. The new derivative, designated CL2A, retains thepH-sensitive carbonate linkage to the SN-38, but it is no longerselectively cleaved by cathepsin B. Nevertheless, it has identical serumstability and improved in vivo activity compared to the original CL2linker (Cardillo et al., 2011, Clin Cancer Res 17:3157-69). Becausesignificant efficacy without toxicity was found with the slowlyinternalizing anti-CEACAM5-SN-38, we postulated that its activity wasaided by the slow release of SN-38 from the antibody after it localizedin a tumor. Thus, the main objective in this report was to evaluate thetherapeutic prospects of conjugates prepared using the CL2A linker withtwo antibodies that are highly specific for B-cell cancers but differ intheir antigen expression and internalization properties.

Epratuzumab (Emab) is a rapidly internalizing (e.g., ≥50% within 1hour), humanized anti-CD22 IgG1 that has been evaluated extensively inlymphoma and leukemia in an unconjugated or conjugated form. Veltuzumab(Vmab) is a humanized anti-CD20 antibody that is also being studiedclinically but internalizes slowly (e.g., ˜10% in 1 hour). CD20 isusually expressed at much higher levels than CD22 in non-Hodgkinlymphoma, whereas CD22 is preferentially expressed in acutelymphoblastic leukemia (ALL) but not in multiple myeloma. Bothantibodies are effective in patients as unconjugated agents, but onlyveltuzumab is active in murine xenograft models (Stein et al., 2004,Clin Cancer Res 10:2868-76). On the basis of previous studies thatshowed ⁹⁰Y-Emab combined with unconjugated veltuzumab had enhancedefficacy in NHL models (Mattes et al., 2008, Clin Cancer Res14:6154-60), we also examined the Emab-SN-38+Vmab combination, as thiscould provide additional benefit without competing for the same targetantigen or having additional toxicity.

Materials and Methods

Cell lines. Ramos, Raji, Daudi (Burkitt lymphomas), and JeKo-1 (mantlecell lymphoma) were purchased from American Type Culture Collection.REH, RS4; 11, MN-60, and 697 (ALL) were purchased from Deutsche Sammlungvon Mikroorganismen and Zellkulturen. WSU-FSCCL (follicular NHL) was thegift of Dr. Mitchell R. Smith (Fox Chase Cancer Center, Philadelphia,Pa.). All cell lines were cultured in a humidified CO₂ incubator (5%) at37° C. in recommended supplemented media containing 10 to 20% fetal calfserum and were checked periodically for Mycoplasma.

Antibodies and conjugation methods. Epratuzumab and veltuzumab arehumanized anti-CD22 and anti-CD20 IgG1 monoclonal antibodies,respectively. Labetuzumab (Lmab), a humanized anti-CEACAM5 IgG1, andRS7, a humanized anti-Trop-2 antibody (both from Immunomedics, Inc.),were used as nonbinding, irrelevant controls. Herein, Emab-SN-38,Vmab-SN-38, and Lmab-SN-38 refer to conjugates prepared using the CL2Alinker that was described above. In vitro studies in human serum showedthat approximately 50% of the active SN-38 moiety is released from theIgG each day (Cardillo et al., 2011, Clin Cancer Res 17:3157-69).Another linker, designated CL2E, is stable in human serum over 14 days,but it contains a cathepsin B cleavage site to facilitate the release ofSN-38 when processed in lysosomes. The method to prepare CL2E and thestructures of the CL2A and CL2E linkers are given in the Examples above.The conjugates contained approximately 6 SN-38 units per IgG (e.g., 1.0mg of the IgG-SN-38 conjugate contains ˜16 μg of SN-38).

In vitro cell binding and cytotoxicity. Flow cytometry was carried outusing the unconjugated specific and irrelevant antibodies incubated for1 hour at 4° C., with binding revealed using fluorescein isothiocyanate(FITC)-Fcγ fragment-specific goat anti-human IgG (JacksonImmunoResearch), also incubated for 1 hour at 4° C. Median fluorescencewas determined on a FACSCALIBUR® flow cytometer (Becton Dickinson) usinga CellQuest software package.

Cytotoxicity was determined using the MTS dye reduction assay (Promega).Dose-response curves [with/without goat anti-human Fcγ F(ab′)₂; JacksonImmunoResearch] were generated from the mean of triplicatedeterminations, and IC₅₀-values were calculated using PRISM® GraphPadsoftware (v5), with statistical comparisons using an F test on the bestfit curves for the data. Significance was set at P<0.05.

Immunoblotting. After 24- or 48-hour exposure to the test agents,markers of early (p21 expression) and late (PARP cleavage) apoptosiswere revealed by Western blotting.

In vivo studies. The subcutaneous Ramos model was initiated byimplanting 1×10⁷ cells (0.2 mL) from culture (>95% viability) into 4- to6-week-old female nude mice (Taconic). Three weeks from implantation,animals with tumors ranging from 0.4 to 0.8 cm³ (measured by caliper,L×W×D) were segregated into groups of animals, each with the same rangeof tumor sizes. Tumor size and body weights were measured at least onceweekly, with animals removed from the study when tumors grew to 3.0 cm³or if they experienced 20% or greater body weight loss. The intravenousWSU-FSCCL and 697 models were initiated by intravenous injection of2.5×10⁶ and 1×10⁷ cells, respectively, in female severe combinedimmunodeficient (SCID) mice (Taconic). Treatment began 5 days afteradministration of the WSU-FSCCL cells and 7 days after the 697inoculation. Animals were observed daily, using hind leg paralysis orother signs of morbidity as surrogate survival endpoints. All treatmentswere given intraperitoneally in ≤0.2 mL. The specific dosages andfrequency are given in the Results section. Because mice convertirinotecan to SN-38 efficiently, irinotecan dosing was adjusted on thebasis of SN-38 equivalents; SN-38 mole equivalents are based on 1.6% ofADC mass and 60% of irinotecan mass.

Efficacy was expressed in a Kaplan-Meier curve, using time toprogression (TTP) as surrogate survival endpoints as indicated above.Statistical analysis was conducted by a log-rank test using PRISM®GraphPad software (significance, P<0.05).

Results

Antigen expression and cytotoxicity in vitro. All cell lines were highlysusceptible to SN-38, with EC₅₀ values ranging from 0.13 nmol/L forDaudi to 2.28 nmol/L for RS4; 11 (Table 14). Except for 697 and RS4; 11,the Emab-SN-38 anti-CD22 conjugate was 2- to 7-fold less effective thanSN-38. This is a common finding with our targeted, as well as othernontargeted, SN-38 conjugates. Despite differences in antigenexpression, the Emab-SN-38 and Vmab-SN-38 had similar potencies as thenonbinding, Lmab-SN-38 anti-CEACAM5 conjugate, which was likely due todissociation of approximately 90% of SN-38 during the 4-day MTS assay.Other in vitro procedures using shorter exposure times were alsoineffective in discriminating differences in the potencies ofconjugates. For example, Annexin V staining after a 1-day exposurefailed to find differences between untreated and treated cells (notshown). Upregulation of p21 and PARP cleavage was also examined as earlyand late markers of apoptosis, respectively. Ramos did not express p21.However, PARP cleavage was detected, but only after a 48-hour exposure,being more strongly expressed in SN-38-treated cells (not shown). TheWSU-FSCCL cell line expressed p21, but neither p21 upregulation nor PARPcleavage was evident until 48 hours after Emab-SN-38 exposure. However,both were observed after a 24-hour exposure with free SN-38 (not shown).While the enhanced intensity and earlier activation of apoptotic eventswith free SN-38 are consistent with its lower EC₅₀ over theIgG-conjugated form, the results indicated that an exposure period of atleast 48 hours would be required, but at this time, approximately 75% ofthe SN-38 would be released from the conjugate.

TABLE 14 Expression of CD20 and CD22 by FACScan and in vitrocytotoxicity by MTS assay of SN-38 and specific Emab anti-CD22-SN-38,Vmab anti-CD20-SN-38, and Lmab anti-CEACAM5-SN-38 conjugates againstseveral hematopoietic tumor cell lines CD20 CD22 expression expressionEC₅₀ values ^(a) Median Median Emab- Vmab- Lmab- Cell fluorescencefluorescence SN-38 95% SN-38, 95% SN-38, 95% SN-38, 95% line(background) (background) nmol/L CI nmol/L CI nmol/L CI nmol/L CINHL:Burkitt Raji 422.2 (6.8) 45.9 (6.8) 1.42 0.8-2.4 2.10 1.2-3.8 ND —ND — 4.61 2.2-9.5 4.88 2.7-9.0 3.73 1.8-7.6 Ramos 620.4 (4.1) 40.8 (4.1)0.40 0.2-0.7 2.92 1.6-5.4 ND — ND — 9.84 4.5-21.6 13.56 4.9-37.2 8.082.9-22.2 Daudi 815.1 (5.9) 145.0 (5.9) 0.13 0.1-0.2 0.52 0.4-0.7 ND — ND— NHL:follicular WSU-FSCCL 97.4 (4.9) 7.7 (4.9) 0.50 0.3-1.0 0.680.4-1.1 ND — ND — 1.05 0.8-1.4 0.83 0.6-1.1 1.17 0.8-1.7 NHL:mantle cellJeko-1 604.6 (6.5) 11.2 (6.5) ND — 2.25 1.3-3.8 1.98 1.1-3.5 2.271.3-3.9 ALL:B cell REH 12.3 (4.1) 22.9 (4.1) 0.47 0.3-0.9 1.22 0.8-1.9ND — ND — 697 6.9 (4.2) 16.0 (4.2) 2.23 1.3-3.9 2.67 1.7-3.7 ND — ND —RS4; 11 3.7 (4.1) 23.3 (4.1) 2.28 1.1-4.9 1.68 1.0-3.0 ND — ND — MN-6021.5 (5.8) 10.3 (5.8) 1.23 0.6-2.1 3.65 2.2-6.2 ND — ND — Abbreviations:CI, confidence interval; ND, not determined. ^(a)EC₅₀ expressed as moleequivalents of SN-38 in Emab-SN-38.

We again examined PARP cleavage and p21 expression, this time in cellstreated with Emab-SN-38+Vmab. Confirming the earlier study in Ramos,PARP cleavage first occurs only after a 48-hour exposure to theconjugate, with expression unchanged in the presence of a cross-linkingantibody (not shown). Exposure to veltuzumab for more than 48 hours hadno effect on PARP cleavage, but cleavage was strong within 24 hours whena cross-linking antibody was added (not shown). However, when veltuzumabalone (no cross-linker) was combined with Emab-SN-38, PARP cleavageoccurred after a 24-hour exposure (not shown), indicating veltuzumabcould induce a more rapid onset of apoptosis, even in the absence ofcross-linking. The only notable difference in the WSU-FSCCL cell linewas that the combination greatly enhanced p21 expression at 48 hours(not shown), again suggesting an acceleration of apoptosis inductionwhen veltuzumab is combined with the Emab-SN-38 conjugate. The delay inapoptosis induction in WSU-FSCCL as compared with Ramos is likelyexplained by the lower expression of CD22 and CD20.

Ultratoxic agents often use linkers that are highly stable in serum, astheir premature release would increase toxicity, but these conjugatesmust be internalized for the drug to be delivered optimally. Becauseepratuzumab internalizes rapidly, we examined whether it might benefitfrom a more stably linked SN-38, comparing in vitro cytotoxicity of theCL2A-linked Emab-SN-38 conjugate with the serum-stable CL2E-SN-38conjugate. Both conjugates had a similar binding affinity (not shown),but the more stable Emab-CL2E-SN-38 was approximately 40- to 55-timesless potent than the CL2A conjugate in 3 cell lines (not shown). Whilespecificity was lacking with the CL2A conjugates, the Emab-CL2E-SN-38consistently was approximately two times more potent than the nonbindingLmab-anti-CEACAM5-CL2E-SN-38 conjugate (not shown). We concluded that itwas unlikely that the more stably linked conjugate would be appropriatefor a slowly internalizing veltuzumab conjugate and therefore continuedour investigation only with CL2A-linked SN-38 conjugates.

Because of limitations of the in vitro assays, efficacy was assessed inxenograft models. As indicated in Table 14, all of the lymphoma celllines have much higher expression of CD20 than CD22. Daudi had thehighest expression of CD22 and CD20, but it is very sensitive in vivo tounconjugated veltuzumab and in vitro testing revealed the highestsensitivity to SN-38 (Table 14). These properties would likely make itdifficult to assess differences in activity attributed to the SN-38conjugate versus the unconjugated antibody, particularly whenunconjugated epratuzumab is not an effective therapeutic in animals.Because Ramos had been used previously to show an advantage forcombining ⁹⁰Y-Emab with veltuzumab (Mattes et al., 2008, Clin Cancer Res14:6154-60), we elected to start with a comparison of the Emab-SN-38 andVmab-SN-38 conjugates in the Ramos human Burkitt cell line. Despite flowcytometry showing a 15-fold higher expression of CD20 over CD22,immunohistology of Ramos xenografts showed abundant CD22 and CD20, withCD22 seemingly expressed more uniformly than CD20 (not shown).

Ramos xenografts in untreated animals progressed rapidly, reaching the3.0-cm³ termination size from their starting size of 0.4 cm³ within 6days (not shown), and as reported previously, neither veltuzumab norepratuzumab appreciably affected the progression of well-establishedRamos xenografts (Sharkey et al., 2009, J Nucl Med 50:444-53).Consistent with previous findings using other SN-38 conjugates, none ofthe animals treated with a 4-week, twice-weekly, 0.5 mg/dose treatmentregimen had appreciable weight loss. Both conjugates were highlyeffective in controlling tumor growth, with 80% or more of the animalshaving no evidence of tumor by the end of the 4-week treatment (FIG.12A-12D). The 0.25-mg Vmab-SN-38 dose was better at controlling growthover the first 4 weeks, but at 0.5 mg, similar early growth control wasobserved for both conjugates. Thus, despite a 15-fold higher expressionof CD20 than CD22, Emab-SN-38 compared favorably with Vmab-SN-38.Therefore, the remaining studies focused on Emab-SN-38 alone or incombination with unconjugated veltuzumab.

Emab-SN-38 dose-response and specificity. A dose-response relationshipwas seen for the specific Emab-SN-38 and irrelevant Lmab-SN-38conjugates, but Emab-SN-38 had significantly better growth control at 2of the 3 levels tested, and with a strong trend favoring the specificconjugate at the intermediate dose (FIG. 13A-13C). Again, 0.25 mg ofEmab-SN-38 ablated a majority of the tumors; here, 7 of 10 animals weretumor-free at the end of the 12-week monitoring period, with no changein body weight. Animals given irinotecan alone (6.5 μg/dose;approximately the same SN-38 equivalents as 0.25 mg of conjugate) had amedian survival of 1.9 weeks, with 3 of 11 animals tumor-free at the endof the study, which was not significantly different from the 3.45-weekmedian survival for the irrelevant Lmab-SN-38 conjugate (P=0.452; FIG.13C).

In the 697-disseminated leukemia model, the median survival ofsaline-treated animals was just 17 days from tumor inoculation. Animalsgiven unconjugated epratuzumab plus irinotecan (same mole equivalents ofSN-38 as 0.5 mg of the conjugate) had the same median survival, whereasanimals given 0.5 mg of Emab-SN-38 twice weekly starting 7 days fromtumor inoculation survived to 24.5 days, significantly longer thanuntreated animals (P<0.0001) or for unconjugated epratuzumab given withirinotecan (P=0.016). However, Emab-SN-38 was not significantly betterthan the irrelevant conjugate (median survival=22 days; P=0.304), mostlikely reflecting the low expression of CD22 in this cell line.

Emab-SN-38 combined with unconjugated Vmab anti-CD20. We previouslyreported improved responses when ⁹⁰Y-Emab was combined with unconjugatedveltuzumab in the subcutaneous Ramos model (Mattes et al., 2008, ClinCancer Res 14:6154-60) and thus this possibility was examined withEmab-SN-38. In a pilot study, 5 animals bearing subcutaneous Ramostumors averaging approximately 0.3 cm³ were given veltuzumab (0.1 mg),0.1 mg of Emab-SN-38, or Emab-SN-38+Vmab (all agents given twice weeklyfor 4 weeks). The median TTP to 2.0 cm³ was 22, 14, and more than 77days, respectively (veltuzumab vs. Emab-SN-38 alone, P=0.59;Emab-SN-38+Vmab vs. Emab-SN-38, P=0.0145), providing an initialindication that the combination of veltuzumab with Emab-SN-38 improvedthe overall therapeutic response. In a follow-up study that also used atwice-weekly, 4-week treatment regimen, 6 of 11 animals given 0.1 mg ofEmab-SN-38 plus 0.1 mg of veltuzumab had no evidence of tumors 16 weeksfrom the start of treatment, whereas the median survival for animalsreceiving veltuzumab alone or with 0.1 mg of the control Lmab-SN-38 was1.9 and 3.3 weeks, respectively, with 3 of 11 animals being tumor-freeat 16 weeks in each of these groups (not shown). Despite the longermedian TTP and more survivors, no significant differences were foundbetween the groups. Thus, in the Ramos model, which has abundant CD20and moderate levels of CD22, the Emab-SN-38 conjugate given at nontoxicdose levels was not significantly better than unconjugated anti-CD20therapy, but the addition of Emab-SN-38 to unconjugated anti-CD20therapy appeared to improve the response without toxicity. It isimportant to emphasize that the SN-38 conjugates are given at levels farless than their maximum tolerated dose, and therefore these resultsshould not be interpreted that the unconjugated anti-CD20 therapy isequal to that of the Emab-SN-38 conjugate.

Two additional studies were conducted in an intravenous implanted modelusing the WSU-FSCCL follicular NHL cell line that has a low expressionof CD20 and CD22 (not shown). The median survival time forsaline-treated animals was 40 to 42 days from tumor implantation.Irinotecan alone (not shown), given at a dose containing the same SN-38equivalents as 0.3 mg of the ADC, increased the median survival (49 vs.40 days, respectively; P=0.042), but 14 of 15 animals succumbed todisease progression on day 49, the same day the final 4 of 15 animals inthe saline group were eliminated (not shown). Despite its relatively lowCD20 expression, veltuzumab alone (35 μg twice weekly×4 weeks) waseffective in this model. The median survival increased to 91 days in thefirst study, with 2 cures (day 161), and to 77 days in the second, butwith no survivors after 89 days (veltuzumab alone vs. saline-treated,P<0.001 in both studies). Unconjugated epratuzumab (0.3 mg/dose)combined with irinotecan and veltuzumab had the same median survival asveltuzumab alone, suggesting that neither epratuzumab nor irinotecancontributed to the net response.

As expected because of the low CD22 expression by WSU-FSCCL, Emab-SN-38alone was not as effective as in Ramos. At the 0.15-mg dose, nosignificant benefit over the saline group was seen, but at 0.3 mg, themedian survival increased to 63 days, providing a significantimprovement compared with the saline-treated animals (P=0.006). Thesecond study, using 0.3 mg of Emab-SN-38, confirmed an enhanced survivalcompared with the saline group (75 vs. 40 days; P<0.0001). Thespecificity of this response was not apparent in the first study, wherethe median survival of the irrelevant Lmab-SN-38 conjugate andEmab-SN-38 were not different at either 0.15- or 0.3-mg dose levels (42vs. 49 days and 63 vs. 63 days for the Emab-SN-38 vs. anti-CEACAM5-SN-38conjugates at the 2 doses levels, respectively). However, in the secondstudy, the 0.3-mg dose of Emab-SN-38 provided a significantly improvedsurvival over the irrelevant conjugate (75 vs. 49 days; P<0.0001).Again, the difficulty in showing specificity in this model is mostlikely related to low CD22 expression.

Combining the specific Emab-SN-38 with veltuzumab substantiallyincreases survival, with evidence of more robust responses than thecontrol Lmab-SN-38. For example, in the first study, animals treatedwith veltuzumab plus 0.15 or 0.3 mg of the control conjugate had amedian survival of 98 and 91 days, respectively, which was similar tothat of veltuzumab alone (91 days; not shown). However, veltuzumab plus0.15 mg of the specific Emab-SN-38 conjugate increased the mediansurvival to 140 days. While this improvement was not significantlyhigher than veltuzumab alone (P=0.257), when the Emab-SN-38 dose wasincreased to 0.3 mg with veltuzumab, 6 of 10 animals remained alive atthe end of the study, providing a significant survival advantage overthe control conjugate plus veltuzumab (P=0.0002). In a second study, themedian survival of veltuzumab alone was shorter than in the first (77vs. 91 days), yet the median survival for the control conjugate withveltuzumab was again 91 days, which now yielded a significant survivaladvantage over veltuzumab alone (P<0.0001). Combining the specificEmab-SN-38 conjugate with veltuzumab extended the median survival to 126days, which was significantly longer than the median survival of 75 and77 days for Emab-SN-38 and veltuzumab alone, respectively (P<0.0001 foreach). However, in this study, it did not quite meet the requirementsfor a statistical improvement over the combination with controlanti-CEACAM5-SN-38 conjugate (P=0.078).

Discussion

Over the past 10 years, ADCs have made substantial gains in cancertherapy, yet there also have been some setbacks. The gains occurredlargely when investigators chose to examine agents that were too toxicto be used alone, but when coupled to an antibody, these so-calledultratoxics produced substantially improved responses in preclinicaltesting. The recent approval of brentuximab vedotin, an auristatinconjugate, in Hodgkin lymphoma and the clinical success withtrastuzumab-DM1 anti-HER2-maytansine conjugate as a single agent inbreast cancer refractive to unconjugated trastuzumab suggest that theseADCs bearing ultratoxic agents are becoming accepted treatmentmodalities. However, conjugates prepared with agents that are themselvespotent in the picomolar range can have an increased risk for toxicity,as the recent decision to withdraw gemtuzumab ozogamicin, theanti-CD33-calicheamicin conjugate, from the market suggests (Ravandi,2011, J Clin Oncol 29:349-51). Thus, the success of an ADC may depend onidentifying appropriate chemistries to bind the drug and antibodytogether, as well as defining a suitable target that is sufficientlyexpressed to allow an adequate and selective delivery of the cytotoxicagent.

We developed a linker for coupling SN-38 to IgG that allows SN-38 to bereleased slowly from the conjugate in serum (about 50% per day). Withthis linker, an antibody that is slowly internalized could be aneffective therapeutic, perhaps because the conjugate localized to atumor releases a sufficient amount of drug locally, even without beinginternalized. The CL2A linker also was used recently with an antibody toTrop-2 that was reported to be internalized rapidly (Cardillo et al.,2011, Clin Cancer Res 17:3157-69). Thus, it appears that the slowrelease mechanism is beneficial for internalizing and noninternalizingantibodies.

In this report, we expanded our assessment of the CL2A linker bycomparing SN-38 conjugates prepared with epratuzumab, a rapidlyinternalizing anti-CD22 IgG, and veltuzumab, a slowly internalizinganti-CD20 IgG, for the treatment of B-cell malignancies, and we have nowfound combinations with DNA-breaking agents, such as microtubuleinhibitors and PARP inhibitors that show synergistic anti-tumor effects.Prior studies with the murine parent of epratuzumab had indicated thatmost of the antibody internalizes within 1 hour and 50% of CD22 isreexpressed on the cell surface within 5 hours (Shih et al., 1994, Int JCancer 56:538-45). This internalization and reexpression process wouldpermit intracellular delivery that might compensate for lower surfaceexpression of CD22. Because many of the B-cell malignancies express muchmore CD20 than CD22, a conjugate targeting CD20 might deliver more molesof drug by releasing its toxic payload after being localized in thetumor.

In vitro cytotoxicity studies could not discriminate the potency of thespecific conjugates or even an irrelevant conjugate because of therelease of SN-38 from the conjugate into the media. Indeed, SN-38 alonewas somewhat more potent than the conjugates, which may reflect itsaccelerated ability to enter the cell and engage topoisomerase I.Because other studies revealed that the conjugates required a 48-hourexposure before early signs of apoptosis could be seen, we concludedthat in vitro testing would not be able to discriminate the potency ofthese 2 conjugates and therefore resorted to in vivo studies.

In xenograft models, both conjugates had similar antitumor activityagainst Ramos tumors, which flow cytometry had indicated expressednearly 15-fold more CD20 than CD22. This lent support to selecting theEmab anti-CD22-SN-38 conjugate especially because it could be combinedwith unconjugated Vmab anti-CD20 therapy without concern that eitheragent would interfere with the binding of the other agent. Indeed, if ananti-CD20-SN-38 conjugate were used, the total IgG protein dose givenlikely would be below a level typically needed for effectiveunconjugated anti-CD20 antibody treatments, as the dose-limitingtoxicity would be driven by the SN-38 content. Adding more unlabeledanti-CD20 to an anti-CD20-SN-38 conjugate would risk reducing theconjugate's uptake and potentially diminishing its efficacy. However, aswe showed previously in combination studies using radiolabeledepratuzumab with unconjugated veltuzumab, benefit can be derived fromboth agents given at their maximum effective and safe dosages. In vitrostudies showed veltuzumab, even in the absence of cross-linking that isused to enhance signaling, accelerated apoptotic events initiated withEmab-SN-38. Thus, as long as the Emab-SN-38 conjugate was as effectiveas the anti-CD20 conjugate, selecting the Emab-SN-38 conjugate is alogical choice because it allows for a more effective combinationtherapy, even in tumors where one or both of the antigens are low inexpression.

Because most ADCs using ultratoxic drugs are stably linked, we alsotested a serum-stable, but intracellularly cleavable, anti-CD22-SN-38conjugate, but determined it was 40- to 55-fold less potent than theCL2A linker. Others have examined a variety of ultratoxic drugsconjugated to anti-CD20 or anti-CD22 antibodies, finding thatinternalizing conjugates are generally more active, but also observingthat even slowly internalizing antibodies could be effective if thereleased drug penetrated the cell membrane. While the CL2A-type linkermay be appropriate for SN-38, it may not be optimal for a more toxicagent, where even a small, sustained release in the serum would increasetoxicity and compromise the therapeutic window.

Emab-SN-38 was active at a cumulative dose of 0.6 mg in mice bearingRamos (75 μg twice weekly for 4 weeks), which extrapolates to a humandose of just 2.5 mg/kg. Thus, Emab-SN-38 should have an ampletherapeutic window in patients. Furthermore, an effective and safe doseof the anti-Trop-2-SN-38 conjugate was combined with a maximum tolerateddose of a ⁹⁰Y-labeled antibody without an appreciable increase intoxicity but with improved efficacy (Sharkey et al., 2011, Mol CancerTher 10:1072-81). Thus, the safety and efficacy profile of these SN-38antibody conjugates are very favorable for other combination therapies.

Even though irinotecan is not used routinely for the treatment ofhematopoietic cancers, SN-38 was as potent in lymphoma and leukemia celllines as in solid tumors (Cardillo et al., 2011, Clin Cancer Res17:3157-69). In the WSU-FSCCL cell line, the specific and irrelevant IgGconjugates were significantly better than irinotecan, whereas in Ramos,the median TTP with the irrelevant conjugate was longer but notsignificantly better than irinotecan. These results are consistent withother studies that have shown that a nonspecific IgG is an excellentcarrier for drugs and more potent in vivo than free drug or conjugatesprepared with albumin or polyethylene glycol (PEG)-Fc. While thePEG-SN-38 conjugate had significant antitumor effects, it was given atits maximum tolerated amounts, ranging from 10 to 30 mg/kg SN-38equivalents (Sapra et al., 2009, Haematologica 94:1456-9). In contrast,the maximum cumulative dose of SN-38 given over 4 weeks to animalsbearing Ramos was only 1.6 mg/kg (i.e., dosing of 0.25 mg of Emab-SN-38given twice weekly over 4 weeks) and this was nontoxic.

The specific therapeutic activity of Emab-SN-38 appeared to improve incell lines with higher CD22 expression. For example, in Ramos, specifictherapeutic effects of Emab-SN-38 alone were recorded at 2 of the 3different dose levels examined, and a sizeable number of tumors werecompletely ablated. In contrast, in WSU-FSCCL that had about 2.5-foldlower expression of CD22, Emab-SN-38 improved survival significantlycompared with the irrelevant anti-CEACAM5-SN-38 conjugate in 1 of 2studies. However, it is important to emphasize that when used incombination with unconjugated anti-CD20 therapy, Emab-SN-38 amplifiesthe therapeutic response. Thus, the combination of these two treatmentscould augment the response even in situations where CD22 is not highlyexpressed.

In conclusion, using the less-stable CL2A-SN-28 linker, Emabanti-CD22-SN-38 conjugate was equally active at nontoxic doses in vivoas a similar anti-CD20-SN-38 conjugate, despite the fact that CD20expression was more than a log-fold higher than CD22. Therapeuticresponses benefited by the combination of Emab-SN-38 with unconjugatedVmab anti-CD20 therapy, even when CD22 expression was low, suggestingthat the combination therapy could improve responses in a number ofB-cell malignancies when both antigens are present The current studiessuggest that this combination is very potent in diverse lymphoma andleukemia preclinical models, yet appears to have less host toxicity.Also, combinations of this ADC with microtubule and PARP inhibitors canbe synergistic in inhibiting tumor growth and extending survival of thetumor-bearing host. This result is surprising, in that it has beenpreviously reported that the PARP inhibitor iniparib failed to sensitizecancer cells to combination therapy with standard anti-cancer agents,such as cisplatin, gemcitabine or paclitaxel (Bao et al., 2015,Oncotarget [Epub ahead of print, Sep. 22, 2015])

Example 16 Anti-CD74 (Milatuzumab) SN-38 Conjugates for Treatment ofCD74+ Human Cancers

Abstract

CD74 is an attractive target for antibody-drug conjugates (ADC), becauseit internalizes and recycles after antibody binding. CD74 mostly isassociated with hematological cancers, but is expressed also in solidcancers. Therefore, the utility of ADCs prepared with the humanizedanti-CD74 antibody, milatuzumab, for the therapy CD74-expressing solidtumors was examined. Milatuzumab-doxorubicin and two milatuzumab-SN-38conjugates were prepared with cleavable linkers (CL2A and CL2E),differing in their stability in serum and how they release SN-38 in thelysosome. CD74 expression was determined by flow cytometry andimmunohistology. In vitro cytotoxicity and in vivo therapeutic studieswere performed in the human cancer cell lines A-375 (melanoma), HuH-7and Hep-G2 (hepatoma), Capan-1 (pancreatic), and NCI-N87 (gastric), andRaji Burkitt lymphoma. The milatuzumab-SN-38 ADC was compared to SN-38ADCs prepared with anti-Trop-2 and anti-CEACAM6 antibodies in xenograftsexpressing their target antigens.

Milatuzumab-doxorubicin was most effective in the lymphoma model, whilein A-375 and Capan-1, only the milatuzumab-CL2A-SN-38 showed atherapeutic benefit. Despite much lower surface expression of CD74 thanTrop-2 or CEACAM6, milatuzumab-CL2A-SN-38 had similar efficacy inCapan-1 as anti-Trop-2 CL2A-SN-38, but in NCI-N87, the anti-CEACAM6 andanti-Trop-2 conjugates were superior. Studies in 2 hepatoma cell linesat a single dose level showed significant benefit over saline-treatedanimals, but not against an irrelevant IgG conjugate. CD74 is a suitabletarget for ADCs in some solid tumor xenografts, with efficacy largelyinfluenced by uniformity of CD74 expression, and with CL2A-linked SN-38conjugates providing the best therapeutic responses.

Introduction

CD74, referred to as invariant chain or Ii, is a type II transmembraneglycoprotein that associates with HLA-DR and inhibits the binding ofantigenic peptides to the class II antigen presentation structure. Itserves as a chaperone molecule, directing the invariant chain complexesto endosomes and lysosomes, an accessory molecule in the maturation of Bcells, using a pathway mediated by NF-kB, and in T-cell responses viainteractions with CD44 (Naujokas et al., 1993, Cell 74:257-68), and itis a receptor for the pro-inflammatory cytokine, macrophage migrationinhibitory factor (Leng et al., 2003, J Exp Med 197:1467-76), which isinvolved in activating cell proliferation and survival pathways.

In normal human tissues, CD74 is primarily expressed in B cells,monocytes, macrophages, dendritic cells, Langerhans cells, subsets ofactivated T cells, and thymic epithelium (not shown), and it isexpressed in over 90% of B-cell tumors (Burton et al., 2004, Clin CancerRes 10:6606-11; Stein et al., 2004, Blood 104:3705-11). Early studieshad conflicting data on whether CD74 is present on the membrane, in partbecause the antibodies to the invariant chain were specific for thecytoplasmic portion of the molecule, but also because there arerelatively few copies on the surface, and its half-life on the cellsurface is very short. Approximately 80% of the CD74 on the cell surfaceis associated with the MHC II antigen HLA-DR (Roche et al., 1993, PNASUSA 90:8581-85). Using the murine anti-CD74 antibody, LL1, the RajiBurkitt lymphoma cell line was estimated to have 4.8×10⁴ copies/cell,but because of rapid intracellular transit, ˜8 ×10⁶ antibody moleculeswere internalized and catabolized per day (Hansen et al., 1996, BiochemJ 320:293-300). Thus, CD74 internalization is highly dynamic, with theantibody being moved quickly from the surface and unloaded inside thecell, followed by CD74 re-expression on the surface. Fab′internalization occurs just as rapidly as IgG binding, indicating thatbivalent binding is not required. Later studies with a CDR-graftedversion of murine LL1, milatuzumab (hLL1), found that the antibody couldalter B-cell proliferation, migration, and adhesion molecule expression(Stein et al., 2004, Blood 104:3705-11; Qu et al., 2002, Proc Am AssocCancer Res 43:255; Frolich et al., 2012, Arthritis Res Ther 14:R54), butthe exceptional internalization properties of the anti-CD74 antibodymade it an efficient carrier for the intracellular delivery of cancertherapeutics (e.g., Griffiths et al., 2003, Clin Cancer Res 9:6567-71).Based on preclinical efficacy and toxicology results, Phase I clinicaltrials with milatuzumab-doxorubicin in multiple myeloma (Kaufman et al.,2008, ASH Annual Meeting Abstracts, 112:3697), as well as non-Hodgkinlymphoma and chronic lymphocytic leukemia, have been initiated.

Interestingly, CD74 also is expressed in non-hematopoietic cancers, suchas gastric, renal, urinary bladder, non-small cell lung cancers, certainsarcomas, and glioblastoma (e.g., Gold et al., 2010, Int J Clin ExpPathol 4:1-12), and therefore it may be a therapeutic target for solidtumors expressing this antigen. Since a milatuzumab-doxorubicinconjugate was highly active in models of hematological cancers, it was alogical choice for this assessment. However, we recently developedprocedures for coupling the highly potent topoisomerase I inhibitor,SN-38, to antibodies. SN-38 is the active form of irinotecan, whosepharmacology and metabolism are well known. These conjugates havenanomolar potency in solid tumor cell lines, and were found to be activewith antibodies that were not actively internalized. Prior studiesindicated a preference for a linker (CL2A) that allowed SN-38 todissociate from the conjugate in serum with a half-life of ˜1 day,rather than other linkers that were either more or less stable in serum.However, given milatuzumab's exceptional internalization capability, anew linker that is highly stable in serum, but can release SN-38 whentaken into the lysosome, was developed.

The current investigation examines the prospects for using these threemilatuzumab anti-CD74 conjugates, one with doxorubicin, and two SN-38conjugates, for effective therapy primarily against solid tumors.

Materials and Methods

Human tumor cell lines. Raji Burkitt lymphoma, A-375 (melanoma), Capan-1(pancreatic adenocarcinoma), NCI-N87 (gastric carcinoma), Hep-G2hepatoma and MC/CAR myeloma cell lines were purchased from AmericanTissue Culture Collection (Manassas, Va.). HuH-7 hepatoma cell line waspurchased from Japan Health Science Research Resources Bank (Osaka,Japan). All cell lines were cultured in a humidified CO₂ incubator (5%)at 37° C. in recommended media containing 10% to 20% fetal-calf serumand supplements. Cells were passaged <50 times and checked regularly formycoplasma.

Antibodies and conjugation methods. Milatuzumab (anti-CD74 MAb),epratuzumab (anti-CD22), veltuzumab (anti-CD20), labetuzumab(anti-CEACAM5), hMN15 (anti-CEACAM6), and hRS7 (anti-Trop-2) arehumanized IgG₁ monoclonal antibodies. CL2A and CL2E linkers and theirSN-38 derivatives were prepared and conjugated to antibodies asdescribed in the Examples above. The milatuzumab-doxorubicin conjugateswere prepared as previously described (Griffiths et al., 2003, ClinCancer Res 9:6567-71). All conjugates were prepared by disulfidereduction of the IgG, followed by reaction with the correspondingmaleimide derivatives of these linkers. Spectrophotometric analysesestimated the drug:IgG molar substitution ratio was 5-7 (1.0 mg of theprotein contains ˜16 μg of SN-38 or 25 μg of doxorubicin equivalent).

In vitro cell binding and cytotoxicity. Assays to compare cell bindingof the unconjugated and conjugated milatuzumab to antigen-positive cellsand cytotoxicity testing used the MTS dye reduction method (Promega,Madison, Wis.).

Flow cytometry and immunohistology. Flow cytometry was performed in amanner that provided an assessment of only membrane-bound or membraneand cytoplasmic antigen. Immunohistology was performed onformalin-fixed, paraffin-embedded sections of subcutaneous tumorxenografts, staining without antigen retrieval methods, using antibodiesat 10 μg/mL that were revealed with an anti-human IgG conjugate.

In vivo studies. Female nude mice (4-8 weeks old) or female SCID mice (7weeks old) were purchased from Taconic (Germantown, N.Y.) and used aftera 1-week quarantine. All agents, including saline controls, wereadministered intraperitoneally twice-weekly for 4 weeks. Specific dosesare given in Results. Toxicity was assessed by weekly weightmeasurements. For the Raji Burkitt lymphoma model, SCID mice wereinjected intravenously with 2.5×10⁶Raji cells in 0.1 mL media. Five dayslater, animals received a single intravenous injection (0.1 mL) of theconjugate or saline (N=10/group). Mice were observed daily for signs ofdistress and paralysis, and were euthanized when either hind-limbparalysis developed, >15% loss of initial weight, or if otherwisemoribund (surrogate survival endpoints).

Subcutaneous tumors were measure by caliper in two dimensions, and thetumor volume (TV) calculated as L×w²/2, where L is the longest diameterand w is the shortest. Measurements were made at least once weekly, withanimals terminated when tumors grew to 1.0 cm³ (i.e., surrogate survivalend-point). The A-375 melanoma cell line (6×10⁶ cells in 0.2 mL) wasimplanted in nude mice and therapy was initiated when tumors averaged0.23±0.06 cm³ (N=8/group). Capan-1 was implanted subcutaneously in nudemice using a combination of tumor suspension from serially-passagedtumors (0.3 mL of a 15% w/v tumor suspension) combined with 8×10⁶ cellsfrom tissue culture. Treatments were initiated when TV averaged0.27±0.05 cm³ (N=10/group). NCI-N87 gastric tumor xenografts wereinitiated by injecting 0.2 mL of a 1:1 (v/v) mixture of matrigel and1×10⁷ cells from terminal culture subcutaneously. Therapy was startedwhen the TV averaged 0.249±0.045 cm³ (N=7/group). The same procedure wasfollowed for developing the Hep-G2 and HuH-7 hepatoma xenografts in nudemice. Therapy was started when Hep-G2 averaged 0.364±0.062 cm³(N=5/group) and HuH-7 averaged 0.298±0.055 cm³ (N=5/group).

Efficacy is expressed in Kaplan-Meier survival curves, using thesurrogate end-points mentioned above for determining the median survivaltimes. Analysis was performed by a log-rank (Mantel-Cox) test usingPrism GraphPad software (LaJolla, Calif.), with significance at P<0.05.

Results

CD74 expression in human tumor cell lines and xenografts. Six cell linesderived from 4 different solid tumor types were identified asCD74-positive based primarily on the analysis of permeabilized cells(Table 15), since the MFI of membrane-only CD74 in the solid tumor celllines very often was <2-fold higher than the background MFI (exceptA-375 melanoma cell line). Surface CD74 expression in Raji was >5-foldhigher than the solid tumor cell lines, but total CD74 in permeabilizedRaji cells was similar to most of the solid tumor cell lines.

TABLE 15 CD74 expression by flow cytometry expressed as mean fluorescentintensity (MFI) of milatuzumab-positive gated cells. Surface and Surfacecytoplasmic hLL1 MFI Ratio hLL1 MFI Ratio Cell line (bkgd)^(a) hLL1:bkgd(bkgd)^(b) hLL1:bkgd Pane CA^(c) Capan-1  22 (12) 1.8 248 (5) 49.6Gastric Hs746T 17 (8) 2.1 144 (5) 28.8 NCI-N87  5 (4) 1.3 220 (6) 36.7Melanoma A-375 16 (3) 5.3 185 (6) 30.8 Hepatoma Hep-G2  9 (6) 1.5 156(5) 31.2 HuH-7  8 (5) 1.6 114 (4) 28.5 Lymphoma Raji 59 (3) 19.6  143(5) 28.6 ND, not done ^(a)Background MFI of cells incubated withGAH-FITC only.

Immunohistology showed Raji subcutaneous xenografts had a largelyuniform and intense staining, with prominent cell surface labeling (notshown). The Hep-G2 hepatoma cell line had the most uniform uptake of thesolid tumors, with moderately strong, but predominantly cytoplasmic,staining (not shown), followed by the A-375 melanoma cell line that hadsomewhat less uniform staining with more intense, yet mostlycytoplasmic, expression (not shown). The Capan-1 pancreatic (not shown)and NCI-N87 (not shown) gastric carcinoma cell lines had moderate(Capan-1) to intense (NCI-N87) CD74 staining, but it was not uniformlydistributed. The HuH-7 hepatoma cell line (not shown) had the leastuniform and the weakest staining.

Immunoreactivity of the conjugates. K_(d) values for unconjugatedmilatuzumab, milatuzumab-CL2A- and CL2E-SN-38 conjugates were notsignificantly different, averaging 0.77 nM, 0.59 nM, and 0.80 nM,respectively. K_(d) values for the unconjugated anddoxorubicin-conjugated milatuzumab measured in the MC/CAR multiplemyeloma cell line were 0.5±0.02 nM and 0.8±0.2 nM, respectively (Sapraet al., 2008, Clin Cancer Res 14:1888-96).

In vitro drug release and serum stabilities of conjugates. The releasemechanisms of SN-38 from the mercaptoethanol-capped CL2A and CL2Elinkers were determined in an environment partially simulating lysosomalconditions, namely, low pH (pH 5.0), and in the presence or absence ofcathepsin B. The CL2E-SN-38 substrate was inert at pH 5 in the absenceof the enzyme (not shown), but in the presence of cathepsin B, cleavageat the Phe-Lys site proceeded quickly, with a half-life of 34 min (notshown). The formation of active SN-38 requires intramolecularcyclization of the carbamate bond at the 10^(th) position of SN-38,which occurred more slowly, with a half-life of 10.7 h (not shown).

As expected, cathepsin B had no effect on the release of active SN-38 inthe CL2A linker. However, CL2A has a cleavable benzyl carbonate bond,releasing active SN-38 at a rate similar to the CL2E linker at pH 5.0,with a half-life of ˜10.2 h (not shown). The milatuzumab-doxorubicinconjugate, which has a pH-sensitive acylhydrazone bond, had a half-lifeof 7 to 8 h at pH 5.0 (not shown).

While all of these linkers release the drug at relatively similar ratesunder lysosomally-relevant conditions, they have very differentstabilities in serum. Milatuzumab-CL2A-SN-38 released 50% of free SN-38in 21.55±0.17 h (not shown), consistent with other CL2A-SN-38conjugates. The CL2E-SN-38 conjugate, however, was highly inert, with ahalf-life extrapolated to 2100 h. The milatuzumab-doxorubicin conjugatereleased 50% of the doxorubicin in 98 h, which was similar to 2 otherantibody-doxorubicin conjugates (not shown).

Cytotoxicity. A significant issue related to the evaluation of theseconjugates was the relative potency of free doxorubicin and SN-38 inhematopoietic and solid tumor cell lines. Our group previously reportedthat SN-38 was active in several B-cell lymphoma and acute leukemia celllines, with potencies ranging from 0.13 to 2.28 nM (Sharkey et al.,2011, Mol Cancer Ther 11:224-34). SN-38 potency in 4 of the solid tumorcell lines that were later used for in vivo therapy studies ranged from2.0 to 6 nM (not shown). Doxorubicin had a mixed response, with 3-4 nMpotency in the Raji lymphoma and the A-375 melanoma cell lines, but itwas nearly 10 times less potent against Capan-1, NCI-N87, and Hep G2cell lines. Other studies comparing the potency of SN-38 to doxorubicinfound: LS174T colon cancer, 18 vs. 18 (nM potency of SN-38 vs.doxorubicin, respectively); MDA-MB-231 breast cancer, 2 vs. 2 nM;SK-OV-4 ovarian cancer, 18 vs. 90 nM; Calu-3 lung adenocarcinoma, 32 vs.582 nM; Capan-2 pancreatic cancer, 37 vs. 221 nM; and NCI-H466 smallcell lung cancer, 0.1 vs. 2 nM. Thus, SN-38 was 5- to 20-fold morepotent than doxorubicin in 4 of these 6 cell lines, with similar potencyin LS174T and MDA-MB-231. Collectively, these data indicate thatdoxorubicin is less effective against solid tumors than SN-38, whileSN-38 appears to be equally effective in solid and hematopoietic tumors.

As expected, the 3 conjugate forms were often some order of magnitudeless potent than the free drug in vitro, since both drugs are expectedto be transported readily into the cells, while drug conjugates requireantibody binding to transport drug inside the cell (not shown). TheCL2A-linked SN-38 conjugate is an exception, since more than 90% of theSN-38 is released from the conjugate into the media over the 4-day assayperiod (Cardillo et al., 2011, Clin Cancer Res 17:3157-69; Sharkey etal., 2011, Mol Cancer Ther 11:224-34). Thus, even if the conjugate wasinternalized rapidly, it would be difficult to discern differencesbetween the free drug and the CL2A-linked drug.

The stable CL2E-linked SN-38 performed comparatively well in the Rajicell line, compared to free SN-38, but it had substantially (7- to16-fold) lower potency in the 4 solid tumor cell lines, suggesting therelatively low surface expression of CD74 may be playing a role inminimizing drug transport in these solid tumors. Themilatuzumab-doxorubicin conjugate had substantial differences in itspotency when compared to the free doxorubicin in all cell lines, whichwas of similar magnitude as the CL2E-SN-38 conjugates to free SN-38 inthe solid tumor cell lines.

In the 6 additional cell lines mentioned above, themilatuzumab-CL2A-SN-38 conjugate was 9- to 60-times more potent than themilatuzumab-doxorubicin conjugate (not shown), but again, this resultwas influenced largely by the fact that the CL2A-linked conjugatereleases most of its SN-38 into the media over the 4-day incubationperiod, whereas the doxorubicin conjugate would at most release 50% ofits drug over this same time. The CL2E-linked milatuzumab was notexamined in these other cell lines.

In vivo therapy of human tumor xenografts. Previous in vivo studies withthe milatuzumab-doxorubicin or SN-38 conjugates prepared with variousantibodies had indicated they were efficacious at doses far lower thantheir maximum tolerated dose (Griffiths et al., 2003, Clin Cancer Res9:6567-71; Sapra et al., 2005, Clin Cancer Res 11:5257-64; Govindan etal., 2009, Clin Cancer Res 15:6052-61; Cardillo et al., 2011, ClinCancer Res 17:3157-69; Sharkey et al., 2011, Mol Cancer Ther 11:224-34),and thus in vivo testing focused on comparing similar, but fixed,amounts of each conjugate at levels that were well-tolerated.

Initial studies first examined the doxorubicin and SN-38 conjugates in adisseminated Raji model of lymphoma in order to gauge how themilatuzumab-doxorubicin conjugate compared to the 2 SN-38 conjugates(not shown). All specific conjugates were significantly better thannon-targeting labetuzumab-SN-38 conjugate or saline-treated animals,which had a median survival of only 20 days (P <0.0001). Despite invitro studies indicating as much as an 8-fold advantage for the SN-38conjugates in Raji, the best survival was seen with themilatuzumab-doxorubicin conjugates, where all animals given a single17.5 mg/kg (350 μg) dose and 7/10 animals given 2.0 mg/kg (40 μg) werealive at the conclusion of the study (day 112) (e.g., 17.5 mg/kg dosemilatuzumab-doxorubicin vs. milatuzumab-CL2A-SN-38, P=0.0012). Survivalwas significantly lower for the more stable CL2E-SN-38 conjugates(P<0.0001 and P=0.0197, 17.5 and 2.0 mg/kg doses for the CL2A vs. CL2E,respectively), even though in vitro studies suggested that bothconjugates would release active SN-38 at similar rates wheninternalized.

Five solid tumor cell lines were examined, starting with the A-375melanoma cell line, since it had the best in vitro response to bothdoxorubicin and SN-38. A-375 xenografts grew rapidly, withsaline-treated control animals having a median survival of only 10.5days (not shown). A 12.5 mg/kg (0.25 mg per animal) twice-weekly dose ofthe milatuzumab-CL2A-SN-38 conjugate extended survival to 28 days(P=0.0006), which was significantly better than the controlepratuzumab-CL2A-SN-38 conjugate having a median survival of 17.5 days(P=0.0089), with the latter not being significantly different from thesaline-treated animals (P=0.1967). The milatuzumab-CL2A conjugateprovided significantly longer survival than the milatuzumab-CL2E-SN-38conjugate (P=0.0014), which had the same median survival of 14 days asits control epratuzumab-CL2E-SN-38 conjugate. Despite giving a 2-foldhigher dose of the milatuzumab-doxorubicin than the SN-38 conjugates,the median survival was no better than the saline-treated animals (10.5days).

As with the A-375 melanoma model, in Capan-1, only the CL2A-linked SN-38conjugate was effective, with a median survival of 35 days,significantly different from untreated animals (P <0.036) (not shown),even at a lower dose (5 mg/kg; 100 μg per animal) (P<0.02). Neither themilatuzumab-CL2E nor the non-targeting epratuzumab-CL2A-SN-38conjugates, or a 2-fold higher dose of the milatuzumab-doxorubicinconjugate, provided any survival advantage (P=0.44 vs. saline). It isnoteworthy that in the same study with animals given the same dose ofthe internalizing anti-Trop-2 CL2A-SN-38 conjugate (hRS7-SN-38;IMMU-132), the median survival was equal to milatuzumab-CL2A-SN-38 (notshown). The hRS7-CL2A-SN-38 conjugate had been identified previously asan ADC of interest for treating a variety of solid tumors (Cardillo etal., 2011, Clin Cancer Res 17:3157-69). The MFI for surface-binding hRS7on Capan-1 was 237 (not shown), compared to 22 for milatuzumab (seeTable 15). Thus, despite having a substantially lower surface antigenexpression, the milatuzumab-CL2A-SN-38 conjugate performed as well asthe hRS7-CL2A-SN-38 conjugate in this model.

With the milatuzumab-doxorubicin conjugate having inferior therapeuticresults in 2 of the solid tumor xenografts, the focus shifted to comparethe milatuzumab-SN-38 conjugates to SN-38 conjugates prepared with otherhumanized antibodies against Trop-2 (hRS7) or CEACAM6 (hMN-15), whichare more highly expressed on the surface of many solid tumors(Blumenthal et al., 2007, BMC Cancer 7:2; Stein et al., 1993, Int JCancer 55:938-46). Three additional xenograft models were examined.

In the gastric tumor model, NCI-N87, animals given 17.5 mg/kg/dose (350μg) of milatuzumab-CL2A-SN-38 provided some improvement in survival, butit failed to meet statistical significance compared to thesaline-treated animals (31 vs. 14 days; P=0.0760) or to the non-bindingveltuzumab anti-CD20-CL2A-SN39 conjugate (21 days; P=0.3128) (notshown). However, the hRS7- and hMN-15-CL2A conjugates significantlyimproved the median survival to 66 and 63 days, respectively (P=0.0001).The MFI for surface-expressed Trop-2 and CEACAM6 were 795 and 1123,respectively, much higher than CD74 that was just 5 (see Table 15).Immunohistology showed a relatively intense cytoplasmic expression ofCD74 in the xenograft of this cell line, but importantly it wasscattered, appearing only in defined pockets within the tumor (notshown). CEACAM6 and Trop-2 were more uniformly expressed than CD74 (notshown), with CEACAM6 being more intensely present both cytoplasmicallyand on the membrane, and Trop-2 primarily found on the membrane. Thus,the improved survival with the anti-CEACAM6 and anti-Trop-2 conjugatesmost likely reflects both higher antigen density and more uniformexpression in NCI-N87.

In the Hep-G2 hepatoma cell line (not shown), immunohistology showed avery uniform expression with moderate cytoplasmic staining of CD74, andflow cytometry indicated a relatively low surface expression (MFI=9).The MFI with hMN-15 was 175 and immunohistology showed a fairly uniformmembrane and cytoplasmic expression of CEACAM6, with isolated pockets ofvery intense membrane staining (not shown). A study in animals bearingHep-G2 xenografts found the milatuzumab-CL2A-SN-38 extended survival to45 days compared to 21 days in the saline-treated group (P=0.0048),while the hMN-15-CL2A-SN-38 conjugate improved survival to 35 days.There was a trend favoring the milatuzumab conjugate overhMN-15-CL2A-SN-38, but it did not achieve statistical significance (46vs. 35 days; P=0.0802). However, the non-binding veltuzumab-CL2A-SN-38conjugate provided a similar survival advantage as the milatuzumabconjugate. We previously observed therapeutic results with non-bindingconjugates could be similar to the specific CL2A-linked conjugate,particularly at higher protein doses, but titration of the specific andcontrol conjugates usually revealed selectively. Thus, neither of thespecific conjugates provided a selective therapeutic advantage at thesedoses in this cell line.

Another study using the HuH-7 hepatoma cell line (not shown), which hadsimilar surface expression, but slightly lower cytoplasmic levels asHep-G2 (see Table 15), found the hMN-15-SN-38 conjugate providing alonger (35 vs.18 days), albeit not significantly different, survivaladvantage than the milatuzumab-CL2A conjugate (P=0.2944). While both thehMN-15 and milatuzumab conjugates were significantly better than thesaline-treated animals (P =0.008 and 0.009, respectively), again,neither conjugate was significantly different from the non-targetedveltuzumab-SN-38 conjugate at this dose level (P=0.4602 and 0.9033,respectively). CEACAM6 surface expression was relatively low in thiscell line (MFI=81), and immunohistology showed that both CD74 (notshown) and CEACAM6 (not shown) were very faint and highly scattered.

Discussion

The antibody-drug conjugate (ADC) approach for tumor-selectivechemotherapy is an area of considerable current interest (e.g., Govindanet al., 2012, Expert Opin Biol Ther 12:873-90; Sapra et al., 2011,Expert Opin Biol Ther 20:1131-49. The recent clinical successes (Pro etal., 2012, Expert Opin Biol Ther 12:1415-21; LoRusso et al., 2011, ClinCancer Res 17:437-47) have occurred in a large part with the adoption ofsupertoxic drugs in place of the conventional chemotherapeutic agentsthat had been used previously. However, target selection, the antibody,and the drug linker are all factors that influence optimal performanceof an ADC. For example, in the case of trastuzumab-DM1, HER2 is abundanton tumors expressing this antigen, the antibody is internalized, and theantibody itself has anti-tumor activity, all of which could combine toenhance therapeutic outcome. In stark contrast, CD74 is expressed at amuch lower level on the surface of cells, but its unique internalizationand surface re-expression properties has allowed a milatuzumab anti-CD74ADC to be effective in hematopoietic cancer xenograft models even with amoderately toxic drug, such as doxorubicin (Griffiths et al., 2003, ClinCancer Res 9:6567-71; Sapra et al., 2005, Clin Cancer Res 11:5257-64).Although doxorubicin is used more frequently in hematopoietic cancers,while SN-38 and other camptothecins are administered to patients withsolid tumors, we decided to assess the utility of doxorubicin and SN-38conjugates of milatuzumab in solid tumors. The milatuzumab-doxorubicinconjugate was effective in xenograft models of various hematologicalcancers, leading to its clinical testing (NCT01101594 and NCT01585688),while several SN-38 conjugates were effective in solid and hematologicaltumor models, leading to 2 new SN-38 conjugates being pursued in Phase Iclinical trials of colorectal and diverse epithelial cancers(NCT01270698 and NCT01631552).

In vitro, unconjugated doxorubicin and SN-38 had similar potency asdoxorubicin against the Raji lymphoma cell line, but SN-38 was morepotent in a number of different solid tumor cell lines. Interestingly,in vivo, the milatuzumab-doxorubicin conjugate provided the bestresponse in Raji as compared to the milatuzumab-SN-38 conjugates.However, in Capan-1 and A-375, milatuzumab-doxorubicin was lesseffective than the CL2A-linked SN-38 milatuzumab conjugate, even thoughin vitro testing had indicated that A-375 was equally sensitive to freedoxorubicin as to free SN-38. Two other cell lines, MDA-MB-231 breastcancer and LS174T colon cancer, also had similar potency with freedoxorubicin as SN-38 in vitro, but since in vitro testing indicatedSN-38 was equally effective in solid and hematological cancers, and withSN-38 having a 5- to 20-fold higher potency than doxorubicin in mostsolid tumor cell lines evaluated, we decided to focus on the 2milatuzumab-SN-38 conjugates for solid tumor therapy. However, to bettergauge the utility of the milatuzumab-SN-38 conjugates, we included acomparative assessment to SN-38 ADCs prepared with antibodies againstother antigens that are present in a variety of solid tumors.

We previously had investigated therapeutic responses with theinternalizing hRS7 anti-Trop-2 CL2A-linked SN-38 conjugate in theCapan-1 cell line (Cardillo et al., 2011, Clin Cancer Res 17:3157-69),and therefore the efficacy of milatuzumab and hRS7 SN-38 conjugates werecompared. In this study, both conjugates significantly improved survivalcompared to control antibodies, with the CL2A-linked SN-38 conjugates ofeach being superior to the CL2E-linked conjugates. Since flow cytometryhad indicated Trop-2 expression was higher than CD74 in Capan-1, thisresult suggested that the transport capabilities of CD74, which wereknown to be exceptional, were more efficient than Trop-2. However, it iswell known that antigen accessibility (i.e., membrane vs. cytoplasm,physiological and “binding-site” barriers) and distribution among cellswithin a tumor are critical factors influencing every form of targetedtherapy, particularly those that depend on adequate intracellulardelivery of a product to individual cells (Thurber et al., 2008, AdvDrug Del Rev 60:1421-34). In situations where the antigen is notuniformly expressed in all cells within the tumor, having a targetedagent that slowly releases its payload after localizing in the tumor,such as the CL2A-linked conjugates, would allow the drug to diffuse tonon-targeted bystander cells, thereby enhancing its efficacy range.Indeed, high antigen expression could potentially impede tumorpenetration as per the binding-site barrier effect, but theextracellular release mechanism could provide a mechanism for the drugto diffuse within the tumor. This mechanism also is thought to aid theefficacy of other conjugates that we have examined using poorlyinternalizing antibodies, such as anti-CEACAM5 and the anti-CEACAM6 usedherein. Conjugates based on milatuzumab rely more heavily on theantibody's direct interaction with the tumor cell, taking advantage ofCD74's rapid internalization and re-expression that can compensate forits lower abundance on the surface of cells. However, this advantagewould be mitigated when CD74 is highly scattered within the tumor, andwithout a mechanism to retain the conjugate within the tumor, thebenefit of the drug's slow release from the conjugate would be lost. Aprevious review of human gastrointestinal tumors by our group suggeststhat they often have a high level of expression with good uniformity(Gold et al., 2010, Int J Clin Exp Pathol 4:1-12).

Example 17 Use of hRS7-SN-38 (IMMU-132) to Treat Therapy-RefractiveMetastatic Breast Cancer

The patient was a 57-year-old woman with stage IV, triple-negative,breast cancer (ET/PR negative, HER-neu negative), originally diagnosedin 2005. She underwent a lumpectomy of her left breast in 2005, followedby Dose-Dense ACT in adjuvant setting in September 2005. She thenreceived radiation therapy, which was completed in November. Localrecurrence of the disease was identified when the patient palpated alump in the contralateral (right) breast in early 2012, and was thentreated with CMF (cyclophosphamide, methotrexate, 5-fluorouracil)chemotherapy. Her disease recurred in the same year, with metastaticlesions in the skin of the chest wall. She then received acarboplatin+TAXOL® chemotherapy regimen, during which thrombocytopeniaresulted. Her disease progressed and she was started on weeklydoxorubicin, which was continued for 6 doses. The skin disease also wasprogressing. An FDG-PET scan on Sep. 26, 2012 showed progression ofdisease on the chest wall and enlarged, solid, axillary nodes. Thepatient was given oxycodone for pain control.

She was given IXEMPRA® from October 2012 until February 2013 (every 2weeks for 4 months), when the chest wall lesion opened up and bled. Shewas then put on XELODA®, which was not tolerated well due to neuropathyin her hands and feet, as well as constipation. The skin lesions wereprogressive and then she was enrolled in the IMMU-132 trial after givinginformed consent. The patient also had a medical history ofhyperthyroidism and visual disturbances, with high risk of CNS disease(however, brain MRI was negative for CNS disease). At the time ofenrollment to this trial, her cutaneous lesions (target) in the rightbreast measured 4.4 cm and 2.0 cm in the largest diameter. She hadanother non-target lesion in the right breast and one enlarged lymphnode each in the right and left axilla.

The first IMMU-132 infusion (12 mg/kg) was started on Mar. 12, 2013,which was tolerated well. Her second infusion was delayed due to Grade 3absolute neutrophil count (ANC) reduction (0.9) on the scheduled day ofinfusion, one week later. After a week delay and after receivingNEULASTA®, her second IMMU-132 was administered, with a 25% dosereduction at 9 mg/kg. Thereafter she has been receiving IMMU-132 onschedule as per protocol, once weekly for 2 weeks, then one week off.Her first response assessment on May 17, 2013, after 3 therapy cycles,showed a 43% decrease in the sum of the long diameter of the targetlesions, constituting a partial response by RECIST criteria. She iscontinuing treatment at the 9 mg/kg dose level. Her overall health andclinical symptoms improved considerably since she started treatment withIMMU-132.

Example 18 Use of hRS7-SN-38 (IMMU-132) to Treat Refractory, Metastatic,Non-Small Cell Lung Cancer

This is a 60-year-old man diagnosed with non-small cell lung cancer. Thepatient is given chemotherapy regimens of carboplatin, bevacizumab for 6months and shows a response, and then after progressing, receivesfurther courses of chemotherapy with carboplatin, etoposide, TAXOTERE®,gemcitabine over the next 2 years, with occasional responses lasting nomore than 2 months. The patient then presents with a left mediastinalmass measuring 6.5×4 cm and pleural effusion.

After signing informed consent, the patient is given IMMU-132 at a doseof 18 mg/kg every other week. During the first two injections, briefperiods of neutropenia and diarrhea are experienced, with 4 bowelmovements within 4 hours, but these resolve or respond to symptomaticmedications within 2 days. After a total of 6 infusions of IMMU-132, CTevaluation of the index lesion shows a 22% reduction, just below apartial response but definite tumor shrinkage. The patient continueswith this therapy for another two months, when a partial response of 45%tumor shrinkage of the sum of the diameters of the index lesion is notedby CT, thus constituting a partial response by RECIST criteria.

Example 19 Use of hRS7-SN-38 (IMMU-132) Plus Olaparib to TreatRefractory, Metastatic, Small-Cell Lung Cancer

This is a 65-year-old woman with a diagnosis of small-cell lung cancer,involving her left lung, mediastinal lymph nodes, and MRI evidence of ametastasis to the left parietal brain lobe. Prior chemotherapy includescarboplatin, etoposide, and topotecan, but with no response noted.Radiation therapy also fails to control her disease. She is then givencombination therapy with IMMU-132 plus olaparib on a 21-day cycle.Olaparib is administered at 200 mg twice a day on days 1-10 of thecycle. IMMU-132 is administered at 8 mg/kg on days 1 and 8 of the cycle.After 3 cycles, there is a 31% shrinkage of sum of the longest diametersof the lung and lymph node tumors by CT, while the putative brainmetastasis is no longer detected. This constitutes a PR by RECIST 1.1,because the shrinkage is confirmed 4 weeks later (35% shrinkage of thesum of all target lesions. She continues her IMMU-132 dosing every 3weeks for another 3 months, and continues to show objective andsubjective improvement of her condition.

Example 20 Therapy of a Gastric Cancer Patient with Stage IV MetastaticDisease with hRS7-SN-38 (IMMU-132) Plus Paclitaxel

This patient is a 60-year-old male with a history of smoking and periodsof excessive alcohol intake over a 40-year-period. He experiences weightloss, eating discomfort and pain not relieved by antacids, frequentabdominal pain, lower back pain, and most recently palpable nodes inboth axilla. He seeks medical advice, and after a workup is shown tohave an adenocarcinoma, including some squamous features, at thegastro-esophageal junction, based on biopsy via a gastroscope.Radiological studies (CT and FDG-PET) also reveal metastatic disease inthe right and left axilla, mediastinal region, lumbar spine, and liver(2 tumors in the right lobe and 1 in the left, all measuring between 2and 4 cm in diameter). His gastric tumor is resected and he is then puton a course of chemotherapy with epirubicin, cisplatin, and5-fluorouracil. After 4 months and a rest period of 6 weeks, he isswitched to docetaxel chemotherapy, which also fails to control hisdisease, based on progression confirmed by CT measurements of themetastatic tumors and some general deterioration.

The patient is then given combination therapy with IMMU-132 (hRS7-SN-38)and paclitaxel on a 21 day cycle. Paclitaxel is administered at a dosageof 175 mg/m² on days 1, 7 and 14 of the cycle. IMMU-132 is administeredat 10 mg/kg on days 1 and 8 of the cycle. After 3 cycles CT studies aredone to assess status of his disease. The infusions are tolerated well,with some mild nausea and diarrhea, and also with Grade 3 neutropenia,controlled with symptomatic medications and with G-CSF (Neulasta®) forthe neutropenia. The CT studies reveal that the sum of his indexmetastatic lesions has decreased by 28%, so he continues on this therapyfor another 5 cycles. Follow-up CT studies show that the disease remainsabout 35% reduced by RECIST criteria from his baseline measurementsprior to combination therapy, and his general condition also appears tohave improved, with the patient regaining an optimistic attitude towardhis disease being under control.

Example 21 Therapy of Advanced Colon Cancer Patient Refractory to PriorChemo-Immunotherapy, Using Only IMMU-130 (Labetuzumab-SN-38)

The patient is a 50-year-old man with a history of stage-IV metastaticcolonic cancer, first diagnosed in 2008 and given a colectomy andpartial hepatectomy for the primary and metastatic colonic cancers,respectively. He then received chemotherapy, as indicated FIG. 14, whichincluded irinotecan, oxaliplatin, FOLFIRINOX (5-fluoruracil, leucovorin,irinotecan, oxaliplatin), and bevacizumab, as well as bevacizumabcombined with 5-fluorouracil/leucovorin, for almost 2 years. Thereafter,he was given courses of cetuximab, either alone or combined with FOLFIRI(leucovorin, 5-flurouracil, irinotecan) chemotherapy during the nextyear or more. In 2009, he received radiofrequency ablation therapy tohis liver metastasis while under chemo-immunotherapy, and in late 2010he underwent a wedge resection of his lung metastases, which wasrepeated a few months later, in early 2011. Despite havingchemo-immunotherapy in 2011, new lung metastases appeared at the end of2011, and in 2012, both lung and liver metastases were visualized. Hisbaseline plasma carcinoembryonic antigen (CEA) titer was 12.5 ng/mL justbefore undergoing the antibody-drug therapy with IMMU-130. The indexlesions chosen by the radiologist for measuring tumor size change bycomputed tomography were the mid-lobe of the right lung and the livermetastases, both totaling 91 mm as the sum of their longest diameters atthe baseline prior to IMMU-130 (anti-CEACAM5-SN-38) therapy.

This patient received doses of 16 mg/kg of IMMU-130 by slow IV infusionevery other week for a total of 17 treatment doses. The patienttolerated the therapy well, having only a grade 1 nausea, diarrhea andfatigue after the first treatment, which occurred after treatments 4 and5, but not thereafter, because he received medication for theseside-effects. After treatment 3, he did show alopecia (grade 2), whichwas present during the subsequent therapy. The nausea, diarrhea, andoccasional vomiting lasted only 2-3 days, and his fatigue after thefirst infusion lasted 2 weeks. Otherwise, the patient tolerated thetherapy well. Because of the long duration of receiving this humanized(CDR-grafted) antibody conjugated with SN-38, his blood was measured foranti-labetuzumab antibody, and none was detected, even after 16 doses.

The first computed tomography (CT) measurements were made after 4treatments, and showed a 28.6% change from the sum of the measurementsmade at baseline, prior to this therapy, in the index lesions. After 8treatments, this reduction became 40.6%, thus constituting a partialremission according to RECIST criteria. This response was maintained foranother 2 months, when his CT measurements indicated that the indexlesions were 31.9% less than the baseline measurements, but somewhathigher than the previous decrease of 40.6% measured. Thus, based oncareful CT measurements of the index lesions in the lung and liver, thispatient, who had failed prior chemotherapy and immunotherapy, includingirinotecan (parent molecule of SN-38), showed an objective response tothe active metabolite of irintotecan (or camptotechin), SN-38, whentargeted via the anti-CEACAM5 humanized antibody, labetuzumab (hMN-14).It was surprising that although irinotecan (CPT-11) acts by releasingSN-38 in vivo, the SN-38 conjugated anti-CEACAM5 antibody provedeffective in a colorectal cancer patient by inducing a partial responseafter the patient earlier failed to respond to his lastirinotecan-containing therapy. The patient's plasma CEA titer reductionalso corroborated the CT findings: it fell from the baseline level of12.6 ng/mL to 2.1 ng/mL after the third therapy dose, and was between1.7 and 3.6 ng/mL between doses 8 and 12. The normal plasma titer of CEAis usually considered to be between 2.5 and 5.0 ng/mL, so this therapyeffected a normalization of his CEA titer in the blood.

Example 22 Therapy of a Patient with Advanced Colonic Cancer withIMMU-130 Plus Eribulin Mesylate

This patient is a 75-year-old woman initially diagnosed with metastaticcolonic cancer (Stage IV). She has a right partial hemicolectomy andresection of her small intestine and then receives FOLFOX,FOLFOX+bevacizumab, FOLFIRI+ramucirumab, and FOLFIRI+cetuximab therapiesfor a year and a half, when she shows progression of disease, withspread of disease to the posterior cul-de-sac, omentum, with ascites inher pelvis and a pleural effusion on the right side of her chest cavity.Her baseline plasma CEA titer just before this therapy is 15 ng/mL. Sheis given combination therapy with eribulin mesylate (1.4 mg/m²) and 10mg/kg IMMU-130 (anti-CEACAM5-SN-38), both administered on days 1 and 8of a 21 day cycle, which is tolerated very well, without any majorhematological or non-hematological toxicities. Within 2 months oftherapy, her plasma CEA titer shrinks modestly to 1.3 ng/mL, but at the8-week evaluation she shows a 21% shrinkage of the index tumor lesions,which increases to a 27% shrinkage at 13 weeks. Surprisingly, thepatient's ascites and pleural effusion both decrease (with the latterdisappearing) at this time, thus improving the patient's overall statusremarkably. The patient continues her investigational therapy.

Example 23 Gastric Cancer Patient with Stage IV Metastatic DiseaseTreated with IMMU-130 Plus Rucaparib

The patient is a 52-year-old male who sought medical attention becauseof gastric discomfort and pain related to eating for about 6 years, andwith weight loss during the past 12 months. Palpation of the stomacharea reveals a firm lump which is then gastroscoped, revealing anulcerous mass at the lower part of his stomach. This is biopsied anddiagnosed as a gastric adenocarcinoma. Laboratory testing reveals nospecific abnormal changes, except that liver function tests, LDH, andplasma CEA are elevated, the latter being 10.2 ng/mL. The patient thenundergoes a total-body PET scan, which discloses, in addition to thegastric tumor, metastatic disease in the left axilla and in the rightlobe of the liver (2 small metastases). The patient has his gastrictumor resected, and then has baseline CT measurements of his metastatictumors. Four weeks after surgery, he receives 3 courses of combinationchemotherapy consisting of a regimen of cisplatin and 5-fluorouracil(CF), but does not tolerate this well, so is switched to treatment withdocetaxel. It appears that the disease is stabilized for about 4 months,based on CT scans, but then the patient's complaints of further weightloss, abdominal pain, loss of appetite, and extreme fatigue causerepeated CT studies, which show increase in size of the metastases by asum of 20% and a suspicious lesion at the site of the original gastricresection.

The patient is then given combination therapy with IMMU-130(anti-CEACAM5-SN-38, 8 mg/kg) and the PARP inhibitor rucaparib (12mg/m²) on a weekly schedule. He tolerates this well, but after 3 weeksshows a grade 3 neutropenia and grade 1 diarrhea. His fourth infusion ispostponed by one week, and then the weekly infusions are reinstituted,with no evidence of diarrhea or neutropenia for the next 4 injections.The patient then undergoes a CT study to measure his metastatic tumorsizes and to view the original area of gastric resection. Theradiologist measures, according to RECIST criteria, a decrease of thesum of the metastatic lesions, compared to baseline prior to combinationtherapy, of 23%. There does not seem to be any clear lesion in the areaof the original gastric resection. The patient's plasma CEA titer atthis time is 7.2 ng/mL, which is much reduced from the baseline value of14.5 ng/mL. The patient continues on weekly combination therapy at thesame dosages, and after a total of 13 infusions, his CT studies showthat one liver metastasis has disappeared and the sum of all metastaticlesions is decreased by 41%, constituting a partial response by RECIST.The patient's general condition improves and he resumes his usualactivities while continuing to receive a maintenance therapy of 8 mg/kgIMMU-130 every third week for another 4 injections. At the lastmeasurement of blood CEA, the value is 4.8 ng/mL, which is within thenormal range for a smoker, which is the case for this patient. HAHAserum measurements do not disclose an anti-antibody or anti-SN-38antibodies, so the therapy appears not to be immunogenic.

Example 24 Therapy of Relapsed Triple-Negative Metastatic Breast Cancerwith IMMU-130 Plus Veliparib

A 58-year-old woman with triple-negative metastatic breast cancerformerly treated with bevacizumab plus paclitaxel, without response,presents with metastases to several ribs, lumbar vertebrae, a solitarylesion measuring 3 cm in diameter in her left lung, with considerablebone pain and fatigue. She is given combination therapy with theanti-CEACAM5 IMMU-130 (hMN-14-SN-48) plus veliparib. IMMU-130 isadministered at 12 mg/kg on days 1 and 8 of a 21-day cycle, whileveliparib is administered once daily at 60 mg. Except for transientgrade 2 neutropenia and some initial diarrhea, she tolerates the therapywell, which is then repeated, after a rest of 2 months, for anothercourse. Radiological examination indicates that she has partial responseby RECIST criteria, because the sum of the diameters of the indexlesions decrease by 39%. Her general condition, including bone pain,also improves, and she returns to almost the same level of activity asprior to her illness.

Example 25 Therapy of Relapsed, Generally Refractive, Metastatic ColonicCarcinoma with hMN-15-SN-38 Plus Paclitaxel

A 46-year-old woman has Stage IV metastatic colonic cancer, with a priorhistory of resection of the primary lesion that also had synchronousliver metastases to both lobes of the liver, as well as a single focusof spread to the right lung; these metastases measured, by CT, between 2and 5 cm in diameter. She undergoes various courses of chemotherapy overa period of 3 years, including 5-fluorouracil, leucovorin, irinotecan,oxaliplatin, cetuximab, and bevacizumab. On two occasions, there isevidence of stabilization of disease or a short-term response, but noreduction of 30% or more of her measured lesions. Her plasma CEA titerat baseline prior to combination therapy is 46 ng/mL, and her totalindex lesions measure a sum of 92 mm.

Combination therapy with hMN-15-SN-38 and paclitaxel is instituted on a21-day cycle. hMN-15-SN-38 is administered at 12 mg/kg on days 1 and 8and paclitaxel at a dosage of 135 mg/m² on days 1, 7 and 14 of thecycle. This cycle is repeated 3 times, with only transient neutropeniaand gastrointestinal side effects (nausea, vomiting, diarrhea).Surprisingly, despite failing to respond to FOLFIRI therapy (whichincludes irinotecan, or CPT-11), the patient shows a partial response byRECIST criteria after completing her therapy. She is then placed on amaintenance schedule of this therapy once every month for the next 6months. Followup scans show that her disease remains under control as apartial response (PR), and the patient is generally in good conditionwith a 90% Karnofsky performance status.

Example 26 Therapy of Relapsed Metastatic Ovarian Cancer with IMMU-130Plus Olaparib

A 66-year-old woman with FIGO stage IV ovarian cancer positive for BRCA1mutatation undergoes primary surgery and postoperative paclitaxel andcarboplatin (TC). After a 20-month platinum-free interval, an elevatedCA125 level and recurrence in the peritoneum is confirmed by CT.Following retreatment with TC, a hypersensitivity reaction occurs to thecarboplatin, which is changed to nedaplatin. A complete response isconfirmed by CT. After an 8-month PFI, an elevated serum CA125 level andrecurrence in the peritoneum and liver are confirmed.

She is then given combination therapy with anti-CEACAM5 IMMU-130(hMN-14-SN-38) plus oliparib. IMMU-130 is administered at 10 mg/kg ondays 1 and 8 of a 21-day cycle, while oliparib is administered at 200 mgtwice a day on days 1-10 of the cycle. Except for transient grade 2neutropenia and some initial diarrhea, she tolerates the therapy well,which is then repeated, after a rest of 2 months, for another course.Radiological examination indicates that she has partial response byRECIST criteria, because the sum of the diameters of the index lesionsdecrease by 45%. Her general condition also improves, and she returns toalmost the same level of activity as prior to her illness.

Example 27 Colonic Cancer Patient with Stage IV Metastatic DiseaseTreated with Anti-CSAp-SN-38 Conjugate Plus Paclitaxel

This patient presents with colonic cancer metastases to the left lobe ofthe liver and to both lungs, after having a resection of a 9-cm sigmoidcolon adenocarinoma, followed by chemo-immunotherapy with FOLIFIRI andcetuximab for 6 months, and then FOLFOX followed by bevacizumab for anadditional period of about 9 months. Ten months after the initialresection and then commencement of therapy, the stable disease thoughtto be present shows progression by the lesions growing and a newmetastasis appearing in the left adrenal gland. Her plasma CEA at thistime is 52 ng/mL, and her general condition appears to havedeteriorated, with abdominal pains, fatigue, and borderline anemia,suggesting possibly internal bleeding.

She is then given combination therapy with an SN-38 conjugate of hMu-9(anti-CSAp) antibody and paclitaxel on a 21 day cycle. The antibody orimmunoconjugate is administered on days 1 and 8 of the cycle at a doseof 12 mg/kg, and paclitaxel is administered at a dosage of 175 mg/m² ondays 1, 7 and 14 of the cycle, which is repeated for additionaltreatment cycles, measuring her blood counts every week and receivingatropine medication to control gastrointestinal reactions. Grade 2alopecia is noted after the first treatment cycle, but only a Grade 1neutropenia. After 3 treatment cycles, her plasma CEA titer is reducedto 19 ng/ml, and at this time her CT measurements show a decrease of theindex lesions in the liver and lungs by 24.1%. After an additional 3courses of therapy, she shows a CT reduction of the index lesions of31.4%, and a decrease in the size of the adrenal mass by about 40%. Thispatient is considered to be responding to combination therapy, andcontinues on this therapy. Her general condition appears to be improved,with less fatigue, no abdominal pain or discomfort, and generally moreenergy.

Example 28 Treatment of Metastatic Pancreatic Cancer withAnti-MUC5ac-SN-38 Immunoconjugate Plus Rucaparib

This 44-year-old patient has a history of metastatic pancreaticcarcinoma, with inoperable pancreas ductal adenocarcinoma in thepancreas head, and showing metastases to left and right lobes of theliver, the former measuring 3×4 cm and the latter measuring 2×3 cm. Thepatient is given a course of gemcitabine but shows no objectiveresponse. Four weeks later, he is given combination therapy withhPAM4-SN-38 i.v. at a dose of 8 mg/kg twice-weekly for 2 weeks, with oneweek off, plus rucaparib (12 mg/m²) once a week. This is repeated foranother 2 cycles. CT studies are done one week later and show a totalreduction in tumor mass (all sites) of 32% (partial response), alongsidea drop in his blood CA19-9 titer from 220 at baseline to 75 at the timeof radiological evaluation. The patient shows only grade 1 nausea andvomiting after each treatment and a grade 2 neutropenia at the end ofthe last treatment cycle, which resolves 4 weeks later. No premedicationfor preventing infusion reactions is given.

Example 29 Use of Anti-CD74 hLL1-SN-38 Plus Oliparib to TreatTherapy-Refractive Metastatic Colonic Cancer (mCRC)

The patient is a 67-year-old man who presents with metastatic coloncancer. Following transverse colectomy shortly after diagnosis, thepatient then receives 4 cycles of FOLFOX chemotherapy in a neoadjuvantsetting prior to partial hepatectomy for removal of metastatic lesionsin the left lobe of the liver. This is followed by an adjuvant FOLFOXregimen for a total of 10 cycles of FOLFOX.

CT shows metastases to liver. His target lesion is a 3.0-cm tumor in theleft lobe of the liver. Non-target lesions included severalhypo-attenuated masses in the liver. Baseline CEA is 685 ng/mL.

The patient is then treated with SN-38 conjugated anti-CD74 milatuzumab(hLL1-SN38) in combination with oliparib. hLL1-SN-38 (10 mg/kg) is givenon days 1 and 8 of a 21-day cycle, while oliparib is administered at 200mg twice a day on days 1-10 of the cycle. The patient experiences nausea(Grade 2) and fatigue (Grade 2) during the first cycle and continues thetreatment without major adverse events. The first response assessmentdone (after 3 cycles) shows shrinkage of the target lesion by 26% bycomputed tomography (CT) and his CEA level decreases to 245 ng/mL. Inthe second response assessment (after 8 cycles), the target lesion hasshrunk by 35%. His overall health and clinical symptoms are considerablyimproved.

Example 30 Treatment of Relapsed Chronic Lymphocytic Leukemia withIMMU-114-SN-38 and Paclitaxel

A 67-year-old man with a history of CLL, as defined by the InternationalWorkshop on Chronic Lymphocytic Leukemia and World Health Organizationclassifications, presents with relapsed disease after prior therapieswith fludarabine, dexamethasone, and rituximab, as well as a regimen ofCVP. He now has fever and night sweats associated with generalized lymphnode enlargement, a reduced hemoglobin and platelet production, as wellas a rapidly rising leukocyte count. His LDH is elevated and thebeta-2-microglobulin is almost twice normal. The patient is giventherapy with anti-HLA-DR IMMU-114-SN-38 (IgG4 hL243-SN-38) conjugate incombination with paclitaxel on a 21-day cycle. The IMMU-114-SN-38 isadministered at 8 mg/kg on days 1 and 8 and paclitaxel is administeredat a dosage of 175 mg/m² on days 1, 7 and 14 of the cycle, then thecycle is repeated. After 4 cycles, evaluation shows that the patient'shematological parameters improve and his circulating CLL cells appear tobe decreasing in number. The therapy is resumed for another 3 cycles,after which his hematological and lab values indicate that he has apartial response.

Example 31 Treatment of Follicular Lymphoma Patient with hA19-SN-38,Rucaparib and Paclitaxel

A 60-year-old male presents with abdominal pain and the presence of apalpable mass. The patient has CT and FDG-PET studies confirming thepresence of the mass with pathologic adenopathies in the mediastinum,axillary, and neck nodes. Lab tests are unremarkable except for elevatedLDH and beta-2-microglobulin. Bone marrow biopsy discloses severalparatrabecular and perivascular lymphoid aggregates. These arelymphocytic with expression of CD20, CD19, and CD10 by immunostaining.The final diagnosis is grade-2 follicular lymphoma, stage IVA, with aFLIPI score of 4. The longest diameter of the largest involved node is 7cm. The patient is given combination therapy with a humanized anti-CD19monoclonal antibody IgG (hA19) conjugated with SN-38, plus rucaparib andpaclitaxel, on a 21-day cycle. The ADC is given at 6 mg/kg on days 7 and14, rucaparib is administered at 10 mg/m² on days 1, 8 and 15, andpaclitaxel is administered at 125 mg/m² on days 1, 7 and 14 of thecycle. After 5 cycles, bone marrow and imaging (CT) evaluations show apartial response, where the measurable lesions decrease by about 60% andthe bone marrow is much less infiltrated. Also, LDH andbeta-2-microglobulin titers also decrease.

Example 32 Treatment of Relapsed Precursor B-Cell ALL with hA20-SN-38Plus Olaparib

This 51-year-old woman has been under therapy for precursor,Philadelphia chromosome-negative, B-cell ALL, which shows the ALL cellsstain for CD19, CD20, CD10, CD38, and CD45. More than 20% of the marrowand blood lymphoblasts express CD19 and CD20. The patient has receivedprior therapy with clofarabine and cytarabine, resulting in considerablehematological toxicity, but no response. A course of high-dosecytarabine (ara-C) was also started, but could not be tolerated by thepatient. She is given hA20-SN-38 (veltuzumab-SN-38) and oliparib on a21-day cycle, with doses of hA20-SN-38 by infusion of 6 mg/kg on days 7and 14 and oliparib at 200 mg twice a day on days 1-10 of the cycle.

After 3 cycles, surprisingly, she shows improvement in her blood andmarrow counts, sufficient for a partial response to be determined. Aftera rest of 2 months because of neutropenia (Grade 3), therapy resumes foranother 4 courses. At this time, she is much improved and is underconsideration for maintenance therapy to try to bring her to a stagewhere she could be a candidate for stem-cell transplantation.

Example 33 Treatment of Lymphoma with Anti-CD22-SN-38 Plus Paclitaxel

The patient is a 62 year-old male with relapsed diffuse large B-celllymphoma (DLBCL). After 6 courses of R-CHOP chemoimmunotherapy, he nowpresents with extensive lymph node spread in the mediastinum, axillary,and inguinal lymph nodes. He is given anti-CD22 epratuzumab-SN-38 pluspaclitaxel on a 21-day cycle. The ADC is administered at a dose of 12mg/kg on days 1 and 7 and paclitaxel is administered at 175 mg/m² ondays 1, 7 and 14 of the cycle. After 3 cycles, the patient is evaluatedby CT imaging, and his total tumor bulk is measured and shows a decreaseof 35% (partial response), which appears to be maintained over the next3 months. Side effects are only thrombocytopenia and grade 1 nausea andvomiting after therapy, which resolve within 2 weeks. No pretherapy forreducing infusion reactions is given.

Example 34 Frontline Therapy of Follicular Lymphoma UsingVeltuzumab-SN-38 and Rucaparib

The patient is a 41-year-old woman presenting with low-grade follicularlymphoma, with measurable bilateral cervical and axillary lymph nodes(2-3 cm each), mediastinal mass of 4 cm diameter, and an enlargedspleen. She is given veltuzumab-SN-38 (anti-CD20-SN-38) in combinationwith rucaparib, with ADC administered at 10 mg/kg on day 1 and rucaparibat 12 mg/m² on days 1, 8 and 15. After 4 cycles, her tumor measurementsby CT show a reduction of 80%. She is then given 2 additional courses oftherapy, and CT measurements indicate that a complete response isachieved. This is confirmed by FDG-PET imaging.

Example 35 Production and Use of Pro-2-Pyrrolinodoxorubicin (P2PDox)

Pro-2-pyrrolinodoxorubicin (P2PDox) was synthesized and conjugated toantibodies as described in U.S. Pat. No. 8,877,202, the Figures andExamples section of which are incorporated herein by reference.Exemplary P2PDox ADCs are disclosed in Table 16 below.

TABLE 16 Exemplary P2PDox-Antibody Conjugates % HPLC Protein P2PDo FreeConjugate Lot recover x/IgG Aggr. drug 1 hIMMU-31- II22-138 75.0% 7.391.9% 0.26% P2PDox 2 hA20-P2PDox II22-135 85.7% 6.79  <2% <0.1% 3hLL1-P2PDox II22-145 88.6% 7.10 2.8%  0.2% 4 hRS7-P2PDox II22-142 80.1%7.17 1.8% 0.12% 5 hMN15- II22-180 74.9% 6.87 1.1% 0.46% P2PDox 6 hMN-14-II22-183 80.2% 6.78 2.1% 0.53% P2PDox

Conjugates were also prepared for hPAM4-P2PDox, hLL2-P2PDox andRFB4-P2PDox, with similar protein recovery and purity (not shown).

In vitro cell-binding studies—Retention of antibody binding wasconfirmed by cell binding assays comparing binding of the conjugate tounconjugated antibody (Chari, 2008, Acc Chem Res 41:98-107). The potencyof the conjugate was tested in a 4-day MTS assay using appropriatetarget cells. The hRS7-P2PDox conjugate exhibited IC₅₀ values of0.35-1.09 nM in gastric (NCI-N87), pancreatic (Capan-1), and breast(MDA-MB-468) human cancer cell lines, with free drug exhibiting0.02-0.07 nM potency in the same cell lines.

Serum stability—Serum stability of prototypical P2PDox conjugate,hRS7-P2PDox, was determined by incubating in human serum at aconcentration of 0.2 mg/mL at 37° C. The incubate was analyzed by HPLCusing butyl hydrophobic interaction chromatography (HIC) column in whichthere was good retention time separation between the peak due to freedrug and that due to conjugate or higher molecular weight species. Thisanalysis showed that there was no release of free drug from theconjugate, suggesting high serum stability of the conjugate. When thesame experiment was repeated with hRS7-doxorubicin conjugate, containingthe same cleavable linker as hRS7-P2PDox, and where the free drug wasindependently verified to be released with a half-life of 96 h, clearformation of free drug peak, namely doxorubicin peak, was seen on HICHPLC.

Surprisingly, it was determined that the P2PDox conjugate was heldtightly to the antibody because it cross-linked the peptide chains ofthe antibody together. The cross-linking stabilizes the attachment ofthe drug to the antibody so that the drug is only releasedintracellularly after the antibody is metabolized. The cross-linkingassists in minimizing toxicity, for example cardiotoxicity, that wouldresult from release of free drug in circulation. Previous use of 2-PDoxpeptide conjugates failed because the drug cross-linked the peptide toother proteins or peptides in vivo. With the present conjugates, theP2PDox is attached to interchain disulfide thiol groups while in theprodrug form. The prodrug protection is rapidly removed in vivo soonafter injection and the resulting 2-PDox portion of the conjugatecross-links the peptide chains of the antibody, forming intramolecularcross-linking within the antibody molecule. This both stabilizes the ADCand prevents cross-linking to other molecules in circulation.

Example 36 In Vivo Studies

General—Tumor size was determined by caliper measurements of length (L)and width (W) with tumor volume calculated as (L×W²)/2. Tumors weremeasured and mice weighed twice a week. Mice were euthanized if theirtumors reached >1 cm³ in size, lost greater than 15% of their startingbody weight, or otherwise became moribund. Statistical analysis for thetumor growth data was based on area under the curve (AUC) and survivaltime. Profiles of individual tumor growth were obtained through linearcurve modeling. An f-test was employed to determine equality of variancebetween groups prior to statistical analysis of growth curves. Atwo-tailed t-test was used to assess statistical significance betweenall the various treatment groups and non-specific controls. For thesaline control analysis a one-tailed t-test was used to assesssignificance. Survival studies were analyzed using Kaplan-Meier plots(log-rank analysis), using the Prism GraphPad Software (v4.03) softwarepackage (Advanced Graphics Software, Inc.; Encinitas, Calif.). All dosesin preclinical experiments are expressed in antibody amounts. In termsof drug, 100 μg of antibody (5 mg/kg) in a 20-g mouse, for example,carries 1.4 μg-2.8 (0.14-0.17 mg/kg) of P2PDox equivalent dose whenusing an ADC with 3-6 drugs/IgG.

A single i.v. dose of ≥300 μg [˜10 μg of P2PDox] of the conjugate waslethal, but 4 doses of 45 μg given in 2 weeks were tolerated by allanimals. Using this dosing regimen, we examined the therapeutic effectof hRS7-P2PDox in 2 human tumor xenograft models, Capan-1 (pancreaticcancer) and NCI-N87 (gastric cancer). Therapy began 7 days after tumortransplantation in nude mice. In the established, 7-day-old, Capan-1model, 100% of established tumors quickly regressed, with no evidence ofre-growth (not shown). This result was reproduced in a repeat experiment(not shown). Similar findings were made in the established NCI-N87 model(not shown), where a 2n^(d) course of therapy, administered after day70, was safely tolerated and led to further regressions of residualtumor (not shown). The internalizing hRS7-SN-38 conjugate, targetingTrop-2, provided better therapeutic responses than a conjugate of apoorly internalizing anti-CEACAM5 antibody, hMN-14 (not shown). Anon-targeted anti-CD20 ADC, hA20-P2PDox, was ineffective, indicatingselective therapeutic efficacy (not shown). Data from a breast cancerxenograft (MDA-MB-468) and a second pancreatic cancer xenograft (notshown) reiterate the same trend of the conjugate's specific andsignificant antitumor effects.

PK and toxicity of hRS7-P2PDox with substitutions of 6.8 or 3.7drug/IgG—Antibody-drug conjugates (ADCs) carrying as much as 8ultratoxic drugs/MAb are known to clear faster than unmodified MAb andto increase off-target toxicity, a finding that has led to the currenttrends to use drug substitutions of ≤4 (Hamblett et al., 2004, ClinCancer Res 10:7063-70). Conjugates were prepared and evaluated with meandrug/MAb substitution ratios (MSRs) of ˜6:1 and ˜3:1. Groups of normalmice (n=5) were administered, i.v., single doses of unmodified hRS7 orhRS7-P2PDox with drug substitution of 6.8 or 3.7 (same protein dose),and serum samples were collected at 30 min, 4 h, 24 h, 72 h, and 168 hpost-injection. These were analyzed by ELISA for antibody concentration(not shown). There were no significant differences in serumconcentrations at various times, indicating that these clearedsimilarly. The PK parameters (Cmax, AUC, etc.) were similar. Conjugateswith either higher or lower drug substitution had similar tolerabilityin nude mice, when the administered at the same dose of conjugated drug.

Therapeutic Efficacy at Minimum Effective Dose (MED)—Anti-TROP-2antibody conjugate, hRS7-P2PDox, was evaluated in nude mice bearingNCI-N87 human gastric cancer xenografts by administering a single bolusprotein dose of 9 mg/kg, 6.75 mg/kg, 4.5 mg/kg, 2.25 mg/kg, or 1 mg/kg.The therapy was started when the mean tumor volume (mTV) was 0.256 cm³.On day 21, mTV in the saline control group (non-treatment group) was0.801±0.181 cm³ which was significantly larger than that in mice treatedwith 9, 6.75, 4.5, or 2.25 mg/kg dose with mTV of 0.211±0.042 cm³,0.239±0.0.054 cm³, 0.264±0.087 cm³, and 0.567±0.179 cm³, respectively(P<0.0047, one tailed t-test). From these, the minimum effective dosewas judged to be 2.25 mg/kg, while 9 mg/kg represented MTD.

MTD of Antibody-P2PDox—An MTD study comparing 2-PDox and P2PDoxconjugates of prototype antibody, hLL1, in mice demonstrated that theP2PDox conjugate was much more potent (not shown). The MTD of a singlei.v. injection was between 100 and 300 μg. The MTD of multipleinjections, at a schedule of every four days for a total of fourinjections (q4dx4) was then determined, using protein doses between 25μg to 150 μg per injection. At these doses, a cumulative dose of between100 and 600 μg was given to the animals. Table 17 below summarizes thevarious groups.

TABLE 17 Dosage and Schedule for MTD of antibody-P2PDox 12 FemaleAthymic Nude Mice Group N Treatment Total Amount 1 3 25 μg i.v. q4dx4100 μg 2 3 50 μg i.v. q4dx4 200 μg 3 3 100 μg i.v. q4dx4  400 μg 4 3 150μg i.v. q4dx4  600 μg

Only those mice treated with 25 μg P2PDox-ADC continue to show no signsof toxicity (not shown). This is a cumulative dose of 100 μg which wasalso the dose tolerated when administered as a single injection (notshown). Therefore, the MTD for multiple injections of a P2PDox-ADC inmice is 25 μg q4dx4 from this experiment. A more detailed analysis ofdata and repetition of the experiment established the MTD forfractionated dosing to be 45 μg of protein dose of the conjugate,administered every 4 days for 2 weeks (45 μg, q4dx4 schedule).

Binding Studies—No significant difference in binding of the antibodymoiety to NCI-N87 gastric carcinoma cells was observed betweenunconjugated hRS7 and P2PDox-hRS7 conjugated to 6 molecules of P2PDoxper antibody (not shown). The lack of effect of conjugation on antibodybinding to target antigen was confirmed for P2PDox-hMN-15(anti-CEACAM6), P2PDox-hLL2 (anti-CD22) and P2PDox-hMN-24 (anti-CEACAM5)conjugates. It is concluded that conjugation of P2PDox to antibodiesdoes not affect antibody-antigen binding activity.

Cytotoxicity Studies—The cytotoxicity of P2PDox-mAb conjugates to targetcells was examined. hRS7-P2PDox and hMN-15-P2PDox were cytotoxic toMDA-MB-468, AG S, NCI-N87 and Capan-1 solid tumor cell lines (notshown). hMN-14-P2PDox was cytotoxic to Capan-1, BxPC-3 and AsPC-1 humanpancreatic tumor lines and AGS, NCI-N87 and LS147T human gastric andcolonic tumor lines (not shown). hLL2-P2PDOx was cytotoxic to Daudi,Raji, Ramos and JVM-3 hematopoietic tumor lines (not shown). IC₅₀ valuesfor the conjugates were in the nanomolar concentration range (notshown).

Example 37 Further In Vivo Studies

Further in vivo efficacy studies were performed in nude mice implantedwith NCI-N87 human gastric cancer xenografts (not shown). One treatmentcycle with 4×45 μg of hRS7-P2PDox rapidly regressed all tumors (notshown). A second treatment cycle was initiated about 2 months after theend of the first cycle, resulting in complete regression of all but oneof the hRS7-P2PDox treated animals. The hA20, hLL1 and hMN-14 conjugateshad little effect on tumor progression (not shown). Administration ofP2PDox-hMN-15 resulted in a delayed regression of gastric cancer, whichwas less effective than the hRS7 conjugate.

The effect of varying dosage schedule on anti-tumor efficacy wasexamined (not shown). The experiment began 9 days after tumorimplantation when mean tumor volume for all groups was 0.383 cm³, andended on day 93 (84 days after initiation of therapy). In this study, asingle dose of 180 two weekly doses of 90 and q4dx4 of 45 μg allresulted in significantly enhanced survival (not shown). For the salinecontrol, 0 of 9 mice survived (not shown). For mice receiving 45 μgq4dx4 of hRS7-P2PDox, 8 of 9 mice were alive at day 94 (not shown). Formice receiving 90 μg weekly×2 of hRS7-P2PDox, 9 of 9 mice were alive atday 94 (not shown). For mice receiving a single dose of 180 μg ofhRS7-P2PDox, 8 of 9 mice were alive at day 94 (not shown). At the samedosage schedule, the control hA20 conjugate had no effect on survival(not shown). A toxicity study showed that the three dosage schedules ofhRS7-P2PDox resulted in similarly low levels of toxicity (not shown).

The hRS7-P2PDox conjugate was also effective in Capan-1 pancreaticcancer (not shown) and was more effective at inhibiting tumor growththan a hRS7-SN-38 conjugate (not shown). The hPAM4-P2PDox conjugate wasalso more effective at inhibiting growth of Capan-1 human pancreaticcancer than an hPAM4-SN-38 conjugate (not shown). At 63 days afterCapan-1 tumor injection (with therapy starting at 1 dayspost-innoculation), 0 of 10 mice were alive in the saline control, 10 of10 mice were alive in mice treated twice weekly×2 weeks with 45 μg ofhPAM4-P2PDox, 2 of 10 mice were alive in mice treated twice weekly×2weeks with 45 μg of hA20-P2PDox, 0 of 10 mice were alive in mice treatedtwice weekly×4 weeks with 250 μg of hPAM4-SN-38, and 0 of 10 mice werealive in mice treated twice weekly×4 weeks with 250 μg of h20-SN-38.

hRS7-P2PDox was substantially more effective than hRS7-SN-38 atinhibiting growth of PxPC-3 pancreatic cancer (not shown) and wasslightly more effective than hRS7-SN-38 at inhibiting growth ofMDA-MB-468 breast cancer (not shown).

The effect of different single doses of hRS7-P2PDox on growth of NCI-N87gastric carcinoma xenografts was examined. Using a single dose, themaximum effect on tumor growth was observed at 90 μg or higher (notshown). A single dose of 45 μg was the minimum required to see asignificant survival benefit compared to saline control (not shown).

The ADCC activity of various hRS7-ADC conjugates was determined incomparison to hRS7 IgG (not shown). PBMCs were purified from bloodpurchased from the Blood Center of New Jersey. A Trop-2-positive humanpancreatic adenocarcinoma cell line (BxPC-3) was used as the target cellline with an effector to target ratio of 100:1. ADCC mediated by hRS7IgG was compared to hRS7-Pro-2-PDox, hRS7-CL2A-SN-38, and the reducedand capped hRS7-NEM. All were used at 33.3 nM. Overall activity was low,but significant (not shown). There was 8.5% specific lysis for the hRS7IgG which was not significantly different from hRS7-Pro-2-PDox. Bothwere significantly better than hLL2 control and hRS7-NEM and hRS7-SN-38(P<0.02, two-tailed t-test). There was no difference between hRS7-NEMand hRS7-SN-38.

Example 38 Treatment of Non-Hodgkin's Lymphoma (NHL) with Anti-CD22P2PDox-Epratuzumab Plus Oliparib

A P2PDox-epratuzumab ADC is prepared as described above. Seventeenpatients with previously untreated or relapsed NHL receive 4 doses of70, 100 or 150 mg P2PDox-epratuzumab injected i.v. on days 1 and 14 of a28 day cycle. Oliparib at 200 mg a day is administered on days 1-7 and14-21 of the cycle. Responses are assessed by CT scans, with otherevaluations including adverse event, B-cell blood levels, serumepratuzumab levels and human anti-epratuzumab (HAHA) titers.

Only occasional, mild to moderate transient injection reactions are seenand no other safety issues except neutropenia up to Grade 3 (butreversible after interrupting therapy until reduced to Grade 1) areobserved. Transient B-cell depletion (up to about 25%) is observed atall dosage levels of P2PDox-epratuzumab in combination with oliparib.The objective response rate (partial responses plus complete responsesplus complete responses unconfirmed) is 47% ( 8/17) with a completeresponse/complete response unconfirmed rate of 24% ( 4/17). Four of theeight objective responses continue for 30 weeks or more. Objectiveresponses are observed at all dose levels of P2PDox-epratuzumab plusoliparib. All serum samples evaluated for human anti-epratuzumabantibody (HAHA) are negative.

Example 39 Treatment of Triple Negative Breast Cancer with P2PDox-hRS7plus paclitaxel

Anti-Trop-2 P2PDox-hRS7 ADC is prepared as described in above. Patientswith triple-negative breast cancer who had failed at least two standardtherapies receive 70 mg P2PDox-hRS7 injected i.v. on day 1 of a 21 daycycle, with paclitaxel administered at 175 mg/m² on days 1, 7 and 14 ofthe cycle. After 3 cycles, objective responses are observed, with anaverage decrease in tumor volume of 35%. All serum samples evaluated forhuman anti-hRS7 antibody (HAHA) are negative.

Example 40 Treatment of Metastatic Colon Cancer with P2PDox-hMN-14 PlusOlaparib

A 52-year old man with metastatic colon cancer (3-5 cm diameters) to hisleft and right liver lobes, as well as a 5 cm metastasis to his rightlung, and an elevated blood CEA value of 130 ng/mL, is treated with thecombination of anti-CEACAM5 hMN-14-P2PDox plus olaparib. A 150 mg doseof hMN-14-P2PDox is administered on days 1 and 14 of a 28 day cycle.Oliparib is administered at 200 mg twice a day on days 1-10 of thecycle. Upon CT evaluation after 2 cycles, a 25% reduction of the totalmean diameters of the 3 target lesions is measured, thus constituting agood stable disease response by RECIST1.1 criteria. At the same time,his blood CEA titer is reduced to 30 ng/mL. Repeated courses of therapycontinue as his neutropenia normalizes.

Example 41 Immunoconjugate Storage

The ADC conjugates are were purified and buffer-exchanged with2-(N-morpholino)ethanesulfonic acid (MES), pH 6.5, and furtherformulated with trehalose (25 mM final concentration) and polysorbate 80(0.01% v/v final concentration), with the final buffer concentrationbecoming 22.25 mM as a result of excipient addition. The formulatedconjugates are lyophilized and stored in sealed vials, with storage at2° C.-8° C. The lyophilized immunoconjugates are stable under thestorage conditions and maintain their physiological activities.

From the foregoing description, one skilled in the art can easilyascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usage andconditions without undue experimentation. All patents, patentapplications and publications cited herein are incorporated byreference.

What is claimed is:
 1. A method of treating cancer, comprising: a) administering to a human patient with HLA-DR positive cancer an immunoconjugate that binds to HLA-DR, wherein the immunoconjugate comprises SN-38 conjugated to an anti-HLA-DR antibody; and b) administering to the patient at least one therapeutic agent selected from the group consisting of a PI3K (phosphoinositide 3-kinase) inhibitor and a Bruton kinase inhibitor.
 2. The method of claim 1, wherein the anti-HLA-DR antibody is a humanized L243 antibody.
 3. The method of claim 1, wherein the SN-38 is conjugated to the anti-HLA-DR antibody via a CL2A linker.
 4. The method of claim 1, wherein the Bruton kinase inhibitor is selected from the group consisting of ibrutinib (PCI-32765), PCI-45292, CC-292 (AVL-292), ONO-4059, GDC-0834, LFM-A13 and RN486.
 5. The method of claim 1, wherein the Bruton kinase inhibitor is ibrutinib.
 6. The method of claim 1, wherein the PI3K inhibitor is selected from the group consisting of idelalisib, Wortmannin, demethoxyviridin, perifosine, PX-866, IPI-145 (duvelisib), BAY 80-6946, BEZ235, RP6530, TGR1202, SF1126, INK1117, GDC-0941, BKM120, XL147, XL765, Palomid 529, GSK1059615, ZSTK474, PWT33597, IC87114, TG100-115, CAL263, PI-103, GNE477, CUDC-907, AEZS-136 and LY294002.
 7. The method of claim 1, wherein the PI3K inhibitor is idelalisib.
 8. The method of claim 1, wherein the patient has failed to respond to at least one other therapy, prior to treatment with the immunoconjugate.
 9. The method of claim 1, wherein the immunoconjugate is administered at a dosage of 6 to 12 mg/kg.
 10. The method of claim 1, wherein the immunoconjugate is administered at a dosage of 8 to 10 mg/kg.
 11. The method of claim 1, wherein the cancer is metastatic.
 12. The method of claim 11, further comprising reducing in size or eliminating the metastases.
 13. The method of claim 1, wherein the cancer is refractory to other therapies but responds to the combination of immunoconjugate and therapeutic agent.
 14. The method of claim 1, wherein the patient has failed to respond to therapy with irinotecan or topotecan, prior to treatment with the immunoconjugate.
 15. The method of claim 1, wherein the antibody is an IgG1 or IgG4 antibody.
 16. The method of claim 1, wherein the antibody has an allotype selected from the group consisting of G1m3, G1m3,1, G1m3,2, G1m3,1,2, nG1m1, nG1m1,2 and Km3 allotypes.
 17. The method of claim 1, further comprising administering to the patient one or more additional therapeutic modalities selected from the group consisting of unconjugated antibodies, radiolabeled antibodies, drug-conjugated antibodies, toxin-conjugated antibodies, gene therapy, chemotherapy, therapeutic peptides, cytokine therapy, oligonucleotides, localized radiation therapy, surgery and interference RNA therapy.
 18. The method of claim 17, wherein the therapeutic modality comprises treatment with an agent selected from the group consisting of 5-fluorouracil, afatinib, aplidin, azaribine, anastrozole, anthracyclines, axitinib, AVL-101, AVL-291, bendamustine, bleomycin, bortezomib, bosutinib, bryostatin-1, busulfan, calicheamycin, camptothecin, carboplatin, 10-hydroxycamptothecin, carmustine, celebrex, chlorambucil, cisplatin (CDDP), Cox-2 inhibitors, irinotecan (CPT-11), SN-38, carboplatin, cladribine, camptothecans, cyclophosphamide, crizotinib, cytarabine, dacarbazine, dasatinib, dinaciclib, docetaxel, dactinomycin, daunorubicin, doxorubicin, 2-pyrrolinodoxorubicine (2P-DOX), cyano-morpholino doxorubicin, doxorubicin glucuronide, epirubicin glucuronide, erlotinib, estramustine, epidophyllotoxin, erlotinib, entinostat, estrogen receptor binding agents, etoposide (VP16), etoposide glucuronide, etoposide phosphate, exemestane, fingolimod, flavopiridol, floxuridine (FUdR), 3′,5′-O-dioleoyl-FudR (FUdR-d0), fludarabine, flutamide, farnesyl-protein transferase inhibitors, fostamatinib, ganetespib, GDC-0834, GS-1101, gefitinib, gemcitabine, hydroxyurea, ibrutinib, idarubicin, idelalisib, ifosfamide, imatinib, L-asparaginase, lapatinib, lenolidamide, leucovorin, LFM-A13, lomustine, mechlorethamine, melphalan, mercaptopurine, 6-mercaptopurine, methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, navelbine, neratinib, nilotinib, nitrosurea, olaparib, plicomycin, procarbazine, paclitaxel, PCI-32765, pentostatin, PSI-341, raloxifene, semustine, sorafenib, streptozocin, SU11248, sunitinib, tamoxifen, temazolomide (an aqueous form of DTIC), transplatinum, thalidomide, thioguanine, thiotepa, teniposide, topotecan, uracil mustard, vatalanib, vinorelbine, vinblastine, vincristine, vinca alkaloids and ZD1839.
 19. The method of claim 1, wherein the cancer is selected from the group consisting of B-cell lymphoma, B-cell leukemia, acute myeloid leukemia, chronic myeloid leukemia, multiple myeloma, cancers of the skin, esophagus, stomach, colon, rectum, pancreas, lung, breast, ovary, bladder, endometrium, cervix, testes, kidney, liver and melanoma.
 20. The method of claim 19, wherein the B-cell leukemia or B-cell lymphoma is selected from the group consisting of indolent forms of B-cell lymphoma, aggressive forms of B-cell lymphoma, chronic lymphocytic leukemia, acute lymphocytic leukemia, hairy cell leukemia, non-Hodgkin's lymphoma, Hodgkin's lymphoma, Burkitt lymphoma, follicular lymphoma, diffuse B-cell lymphoma, mantle cell lymphoma and multiple myeloma. 